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The key features of plant development are that the body plan is indefinite, with continual stem cell activity producing new organs, and that there is an alternation of generations in which the phenotypes of haploid cells are determined mainly by their genotype. These life cycle features allow somatic and gametic selection to operate more stringently than in complex animals with a fixed body plan and in animal gametes. Historically our primary focus has been the regulation of MuDR/Mu transposable elements in the context of the maize life cycle. The transposons switch from "cut and paste" to a net replicative mode of transposition in cells that have acquired pre-meiotic fate. To understand how MuDR/Mu exploit this cell fate specification event, we swtiched to studying cell fate specification in maize anthers to understand the basic biology of this organ.

Plants do not have a germ line. Instead, within each flower a small number of somatic cells must be programmed to adopt a pre-meiotic fate. On the male side, this cell fate specification event occurs in the anthers when pluripotent stem cells become archesporial cells. The anther lobes have just 5 cell types, including the cells that ultimately undergo meiosis. Using a panel of male sterile mutants, transcriptome profiling, proteomics, and genetic analysis we are defining how these archesporial and somatic cells acquire their fates, and then maintain them. We recently discovered that hypoxia, generating a signal mediated by the MSCA1 glutaredoxin, establishes which cells differentiate as pre-meiotic cells and then in turn program the somatic niche surrounding them using a secreted protein. Mobile secreted proteins play key roles in establishing cell fate and programming particular cell division patterns. MAC1 also inhibits archesporial cell division -- either directly or as a consequence of somatic differentiation -- until there is an entire column of such cells in each anther lobe; then the archesporial cells start transit amplifying divisions and a 5 days later start meiosis synchronously.

Using additional mutants and laser capture microdissection we are analyzing the steps in differentiation of individual cell types and investigating whether there are changes in DNA methylation. We are particularly interested in characteristics of the archesporial cells and the neighboring tapetum. Many male sterile mutants have defects in tapetal cell fate specification, commitment, or differentiation, later resulting in meiotic arrest. Our most intriguing finding about the archesporial cells is that as soon as they are specified they being making both the mRNA and proteins utilized in meiosis.

We have intriguing clues that a novel type of small RNA (phasiRNAs = phased small RNAs of 21 or 24 nucleotides) are critical for early steps in in anther development. PHAS loci are non repetitive, transcribed by RNA Pol II but do not encode proteins; the long non-coding transcript is processed into precisely the same 21 or 24 nt pieces by the binding of a 22 nt trigger molecule and the action of a specific Dicers (DCL4 for the 21 nt type and DCL5 for the 24 nt class). Only grass anthers produce the 24 nt phasiRNAs, and in maize they appear shortly before the start of meiosis. Based on current evidence, we hypothesize that epidermal cells make the 21 nt phasiRNAs and the tapetal cells adjacent to the meiotic cells make the 24 nt phasiRNAs. Genetic and molecular approaches are being used to discover the functions of these fascinating small molecules.

Abstract

Successful male gametogenesis involves orchestration of sequential gene regulation for somatic differentiation in pre-meiotic anthers. We report here the cloning of Male Sterile23 (Ms23), encoding an anther-specific predicted basic helix-loop-helix (bHLH) transcription factor required for tapetal differentiation; transcripts localize initially to the precursor secondary parietal cells then predominantly to daughter tapetal cells. In knockout ms23-ref mutant anthers, five instead of the normal four wall layers are observed. Microarray transcript profiling demonstrates a more severe developmental disruption in ms23-ref than in ms32 anthers, which possess a different bHLH defect. RNA-seq and proteomics data together with yeast two-hybrid assays suggest that MS23 along with MS32, bHLH122 and bHLH51 act sequentially as either homo- or heterodimers to choreograph tapetal development. Among them, MS23 is the earliest-acting factor, upstream of bHLH51 and bHLH122, controlling tapetal specification and maturation. By contrast, MS32 is constitutive and independently regulated and is required later than MS23 in tapetal differentiation.

Abstract

In seed plants, anthers are critical for sexual reproduction, because they foster both meiosis and subsequent pollen development of male germinal cells. Male-sterile mutants are analyzed to define steps in anther development. Historically the major topics in these studies are meiotic arrest and post-meiotic gametophyte failure, while relatively few studies focus on pre-meiotic defects of anther somatic cells. Utilizing morphometric analysis we demonstrate that pre-meiotic mutants can be impaired in anticlinal or periclinal cell division patterns and that final cell number in the pre-meiotic anther lobe is independent of cell number changes of individual differentiated somatic cell types. Data derived from microarrays and from cell wall NMR analyses allow us to further refine our understanding of the onset of phenotypes. Collectively the data highlight that even minor deviations from the correct spatiotemporal pattern of somatic cell proliferation can result in male sterility in Zea mays.

Abstract

Research into anther ontogeny has been an active and developing field, transitioning from a strictly lineage-based view of cellular differentiation events to a more complex understanding of cell fate specification. Here we describe the modern interpretation of pre-meiotic anther development, from the earliest cell specifications within the anther lobes through SPL/NZZ-, MSP1-, and MEL1-dependent pathways as well as the initial setup of the abaxial and adaxial axes and outgrowth of the anther lobes. We then continue with a look at the known information regarding further differentiation of the somatic layers of the anther (the epidermis, endothecium, middle layer, and tapetum), with an emphasis on male-sterile mutants identified as defective in somatic cell specification. We also describe the differences in developmental stages among species and use this information to discuss molecular studies that have analyzed transcriptome, proteome, and small-RNA information in the anther.

Abstract

Plants lack a germ line; consequently, during reproduction adult somatic cells within flowers must switch from mitotic proliferation to meiosis. In maize (Zea mays L.) anthers, hypoxic conditions in the developing tassel trigger pre-meiotic competence in the column of pluripotent progenitor cells in the center of anther lobes, and within 24 hr these newly specified germinal cells have patterned their surrounding neighbors to differentiate as the first somatic niche cells. Transcriptomes were analyzed by microarray hybridization in carefully staged whole anthers during initial specification events, after the separation of germinal and somatic lineages, during the subsequent rapid mitotic proliferation phase, and during final pre-meiotic germinal and somatic cell differentiation. Maize anthers exhibit a highly complex transcriptome constituting nearly three-quarters of annotated maize genes, and expression patterns are dynamic. Laser microdissection was applied to begin assigning transcripts to tissue and cell types and for comparison to transcriptomes of mutants defective in cell fate specification. Whole anther proteomes were analyzed at three developmental stages by mass spectrometric peptide sequencing using size-fractionated proteins to evaluate the timing of protein accumulation relative to transcript abundance. New insights include early and sustained expression of meiosis-associated genes (77.5% of well-annotated meiosis genes are constitutively active in 0.15 mm anthers), an extremely large change in transcript abundances and types a few days before meiosis (including a class of 1340 transcripts absent specifically at 0.4 mm), and the relative disparity between transcript abundance and protein abundance at any one developmental stage (based on 1303 protein-to-transcript comparisons).

Abstract

Developmental cues to start meiosis occur late in plants. Ameiotic1 (Am1) encodes a plant-specific nuclear protein (AM1) required for meiotic entry and progression through early prophase I. Pollen mother cells (PMCs) remain mitotic in most am1 mutants including am1-489, while am1-praI permits meiotic entry but PMCs arrest at the leptotene/zygotene (L/Z) transition, defining the roles of AM1 protein in two distinct steps of meiosis. To gain more insights into the roles of AM1 in the transcriptional pre-meiotic and meiotic programs, we report here an in depth analysis of gene expression alterations in carefully staged anthers at 1 mm (meiotic entry) and 1.5 mm (L/Z) caused by each of these am1 alleles.1.0 mm and 1.5 mm anthers of am1-489 and am1-praI were profiled in comparison to fertile siblings on Agilent® 4 × 44 K microarrays. Both am1-489 and am1-praI anthers are cytologically normal at 1.0 mm and show moderate transcriptome alterations. At the 1.5-mm stage both mutants are aberrant cytologically, and show more drastic transcriptome changes. There are substantially more absolute On/Off and twice as many differentially expressed genes (sterile versus fertile) in am1-489 than in am1-praI. At 1.5 mm a total of 4,418 genes are up- or down-regulated in either am1-489 or am1-praI anthers. These are predominantly stage-specific transcripts. Many putative meiosis-related genes were found among them including a small subset of allele-specific, mis-regulated genes specific to the PMCs. Nearly 60% of transcriptome changes in the set of transcripts mis-regulated in both mutants (N = 530) are enriched in PMCs, and only 1% are enriched in the tapetal cell transcriptome. All array data reported herein will be deposited and accessible at MaizeGDB http://www.maizegdb.org/.Our analysis of anther transcriptome modulations by two distinct am1 alleles, am1-489 and am1-praI, redefines the role of AM1 as a modulator of expression of a subset of meiotic genes, important for meiotic progression and provided stage-specific insights into the genetic networks associated with meiotic entry and early prophase I progression.

Abstract

One fundamental difference between plants and animals is the existence of a germ-line in animals and its absence in plants. In flowering plants, the sexual organs (stamens and carpels) are composed almost entirely of somatic cells, a small subset of which switch to meiosis; however, the mechanism of meiotic cell fate acquisition is a long-standing botanical mystery. In the maize (Zea mays) anther microsporangium, the somatic tissues consist of four concentric cell layers that surround and support reproductive cells as they progress through meiosis and pollen maturation. Male sterility, defined as the absence of viable pollen, is a common phenotype in flowering plants, and many male sterile mutants have defects in somatic and reproductive cell fate acquisition. However, without a robust model of anther cell fate acquisition based on careful observation of wild-type anther ontogeny, interpretation of cell fate mutants is limited. To address this, the pattern of cell proliferation, expansion, and differentiation was tracked in three dimensions over 30 days of wild-type (W23) anther development, using anthers stained with propidium iodide (PI) and/or 5-ethynyl-2'-deoxyuridine (EdU) (S-phase label) and imaged by confocal microscopy. The pervading lineage model of anther development claims that new cell layers are generated by coordinated, oriented cell divisions in transient precursor cell types. In reconstructing anther cell division patterns, however, we can only confirm this for the origin of the middle layer (ml) and tapetum, while young anther development appears more complex. We find that each anther cell type undergoes a burst of cell division after specification with a characteristic pattern of both cell expansion and division. Comparisons between two inbreds lines and between ab- and adaxial anther florets indicated near identity: anther development is highly canalized and synchronized. Three classical models of plant organ development are tested and ruled out; however, local clustering of developmental events was identified for several processes, including the first evidence for a direct relationship between the development of ml and tapetal cells. We speculate that small groups of ml and tapetum cells function as a developmental unit dedicated to the development of a single pollen grain.

Abstract

Maize anther ontogeny is complex, with the expression of more than 30,000 genes over 4 days of cell proliferation, cell fate acquisition and the start of meiosis. Although many male-sterile mutants disrupt these key steps, few have been investigated in detail. The terminal phenotypes of Zea mays (maize) male sterile 8 (ms8) are small anthers exhibiting meiotic failure. Here, we document much earlier defects: ms8 epidermal cells are normal in number but fail to elongate, and there are fewer, larger tapetal cells that retain, rather than secrete, their contents. ms8 meiocytes separate early, have extra space between them, occupied by excess callose, and the meiotic dyads abort. Thousands of transcriptome changes occur in ms8, including ectopic activation of genes not expressed in fertile siblings, failure to express some genes, differential expression compared with fertile siblings and about 40% of the differentially expressed transcripts appear precociously. There is a high correlation between mRNA accumulation assessed by microarray hybridization and quantitative real-time reverse transcriptase polymerase chain reaction. Sixty-three differentially expressed proteins were identified after two-dimensional gel electrophoresis followed by liquid chromatography tandem mass spectroscopy, including those involved in metabolism, plasmodesmatal remodeling and cell division. The majority of these were not identified by differential RNA expression, demonstrating the importance of proteomics in defining developmental mutants.

Abstract

Infection of maize by corn smut (Ustilago maydis) provides an agronomically important model of biotrophic host-pathogen interactions. After penetration of the maize epidermis, fungal colonization of host tissue induces tumor formation on all aerial maize organs. We hypothesized that transformation of different primordia into plant tumors would require organ-specific gene expression by both host and pathogen and documented these differences by transcriptome profiling. Phenotypic screening of U. maydis mutants deleted for genes encoding secreted proteins and maize mutants with organ-specific defects confirmed organ-restricted tumorigenesis. This is the foundation for exploring how individual pathogen effectors, deployed in an organ-specific pattern, interact with host factors to reprogram normal ontogeny into a tumor pathway.

Abstract

The biotrophic pathogen Ustilago maydis causes tumors by redirecting vegetative and floral development in maize (Zea mays L.). After fungal injection into immature tassels, tumors were found in all floral organs, with a progression of organ susceptibility that mirrors the sequential location of foci of cell division in developing spikelets. There is sharp demarcation between tumor-forming zones and areas with normal spikelet maturation and pollen shed; within and immediately adjacent to the tumor zone, developing anthers often emerge precociously and exhibit a range of developmental defects suggesting that U. maydis signals and host responses are restricted spatially. Male-sterile maize mutants with defects in anther cell division patterns and cell fate acquisition prior to meiosis formed normal adult leaf tumors, but failed to form anther tumors. Methyl jasmonate and brassinosteroid phenocopied these early-acting anther developmental mutants by generating sterile zones within tassels that never formed tumors. Although auxin, cytokinin, abscisic acid and gibberellin did not impede tassel development, the Dwarf8 mutant defective in gibberellin signaling lacked tassel tumors; the anther ear1 mutant reduced in gibberellin content formed normal tumors; and Knotted1, in which there is excessive growth of leaf tissue, formed much larger vegetative and tassel tumors. We propose the hypothesis that host growth potential and tissue identity modulate the ability of U. maydis to redirect differentiation and induce tumors.

Abstract

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Abstract

Oligonucleotide arrays were used to profile gene expression in dissected maize anthers at four stages: after-anther initiation, at the rapid mitotic proliferation stage, pre-meiosis, and meiotic prophase I. Nearly 9200 sense and antisense transcripts were detected, with the most diverse transcriptome present at the pre-meiotic stage. Three male-sterile mutants lacking a range of normal cell types resulting from a temporal progression of anther failure were compared with fertile siblings at equivalent stages by transcription profiles. The msca1 mutant has the earliest visible phenotype, develops none of the normal anther cell types and exhibits the largest deviation from fertile siblings. The mac1 mutant has an excess of archesporial derivative cells and lacks a tapetum and middle layer, resulting in moderate transcriptional deviations. The ms23 mutant lacks a differentiated tapetum and shows the fewest differences from fertile anthers. By combining the data sets from the comparisons between individual sterile and fertile anthers, candidate genes predicted to play important roles during maize anther development were assigned to stages and to likely cell types. Comparative analyses with a data set of anther-specific genes from rice highlight remarkable quantitative similarities in gene expression between these two grasses.

Abstract

A comparative analysis, by expression profiling of maize, was performed to identify novel components in the mechanisms of maize responses to UV-B. Five high-altitude landraces grown from 2,000 to 3,400 m naturally receive higher UV-B fluence than plants at lower altitudes and similar latitudes. These high-altitude landraces were compared directly with a low-altitude line and with literature reports for other temperate maize lines. A microarray analysis demonstrated that among the UV-B responsive transcripts, several types of gene implicated in chromatin remodeling are differentially expressed before and after UV-B treatment in high-altitude lines. RNAi transgenic plants with lower expression of four such chromatin-associated genes exhibited hypersensitivity to UV-B by measurements of leaf arching, increased leaf chlorosis and necrosis, and altered UV-B regulation of selected genes. These results collectively suggest that genes involved in chromatin remodeling are crucial for UV-B acclimation and that some high-altitude lines exhibit adaptations to this challenge.

Abstract

There are thousands of maize lines with distinctive normal as well as mutant phenotypes. To determine the validity of comparisons among mutants in different lines, we first address the question of how similar the transcriptomes are in three standard lines at four developmental stages.Four tissues (leaves, 1 mm anthers, 1.5 mm anthers, pollen) from one hybrid and one inbred maize line were hybridized with the W23 inbred on Agilent oligonucleotide microarrays with 21,000 elements. Tissue-specific gene expression patterns were documented, with leaves having the most tissue-specific transcripts. Haploid pollen expresses about half as many genes as the other samples. High overlap of gene expression was found between leaves and anthers. Anther and pollen transcript expression showed high conservation among the three lines while leaves had more divergence. Antisense transcripts represented about 6 to 14 percent of total transcriptome by tissue type but were similar across lines. Gene Ontology (GO) annotations were assigned and tabulated. Enrichment in GO terms related to cell-cycle functions was found for the identified antisense transcripts. Microarray results were validated via quantitative real-time PCR and by hybridization to a second oligonucleotide microarray platform.Despite high polymorphisms and structural differences among maize inbred lines, the transcriptomes of the three lines displayed remarkable similarities, especially in both reproductive samples (anther and pollen). We also identified potential stage markers for maize anther development. A large number of antisense transcripts were detected and implicated in important biological functions given the enrichment of particular GO classes.

Unique features of the plant life cycle and their consequencesNATURE REVIEWS GENETICSWalbot, V., Evans, M. M.2003; 4 (5): 369-379

Abstract

Continuous development, the absence of a germline, flexible and reversible cellular differentiation, and the existence of haploid and diploid generations--both of which express genes--are characteristics that distinguish plants from animals. Because these differences alter the impact of mutations, animals and plants experience varied selection pressures. Despite different life-cycles, both flowering plants and multicellular animals have evolved complex sensing mechanisms that act after fertilization as 'quality checks' on reproduction, and that detect chromosome dosage and the parent of origin for specific genes. Although flowering plant embryos escape such surveillance in vitro, embryo success in the seed often depends on a healthy endosperm--a nutritive tissue that is produced by a second fertilization event in which maternal and paternal gene contributions can be monitored immediately after fertilization and throughout development.

Abstract

Homology-dependent gene silencing contributes to genomic stability through suppression of transposable elements. Co-ordinate epigenetic silencing is the main regulatory mechanism controlling dispersed, multicopy MuDR/Mu elements responsible for Mutator activity in maize. Silencing eliminates transposition and proceeds through transcriptional inactivation of MuDR genes and DNA methylation of the terminal inverted repeats (TIRs) in both the regulatory MuDR and non-autonomous Mu elements. In plants with active MuDR/Mu elements, initiation of silencing coincides with nuclear retention of non-polyadenylated RNA derived from MuDR and recently described MuDR homologs (hMuDR elements). Nuclear accumulation of MuDR/hMuDR RNA is developmentally progressive, paralleling loss of Mutator activity and is predictive of loss of Mu somatic excision in the progeny. A high ratio of nuclear to cytoplasmic RNA is the earliest molecular marker for MuDR silencing suggesting that the nuclear RNA may trigger transcriptional silencing. We also demonstrate the constitutive presence of small transposon-specific RNAs of 21-26 nucleotides in all maize lines tested, independent of the Mutator activity. The role of the small RNAs in transposon silencing and translational regulation of transposon-encoded proteins is discussed.

Abstract

CRISPR/Cas9 is a powerful genome editing tool in many organisms, including a number of monocots and dicots. Although the design and application of CRISPR/Cas9 is simpler compared to other nuclease-based genome editing tools, optimization requires the consideration of the DNA delivery and tissue regeneration methods for a particular species to achieve accuracy and efficiency. Here, we describe a public sector system, ISU Maize CRISPR, utilizing Agrobacterium-delivered CRISPR/Cas9 for high-frequency targeted mutagenesis in maize. This system consists of an Escherichia coli cloning vector and an Agrobacterium binary vector. It can be used to clone up to four guide RNAs for single or multiplex gene targeting. We evaluated this system for its mutagenesis frequency and heritability using four maize genes in two duplicated pairs: Argonaute 18 (ZmAgo18a and ZmAgo18b) and dihydroflavonol 4-reductase or anthocyaninless genes (a1 and a4). T0 transgenic events carrying mono- or diallelic mutations of one locus and various combinations of allelic mutations of two loci occurred at rates over 70% mutants per transgenic events in both Hi-II and B104 genotypes. Through genetic segregation, null segregants carrying only the desired mutant alleles without the CRISPR transgene could be generated in T1 progeny. Inheritance of an active CRISPR/Cas9 transgene leads to additional target-specific mutations in subsequent generations. Duplex infection of immature embryos by mixing two individual Agrobacterium strains harbouring different Cas9/gRNA modules can be performed for improved cost efficiency. Together, the findings demonstrate that the ISU Maize CRISPR platform is an effective and robust tool to targeted mutagenesis in maize.

Abstract

Although anthers of Zea mays, Oryza sativa, and Arabidopsis thaliana have been studied intensively using genetic and biochemical analyses in the past 20 years, few updates to anther anatomical and ultrastructural descriptions have been reported. For example, no transmission electron microscopy (TEM) images of the premeiotic maize anther have been published. Here we report the presence of chloroplasts in maize anthers.TEM imaging, electron acceptor photosynthesis assay, in planta photon detection, microarray analysis, and light and fluorescence microscopy were used to investigate the presence of chloroplasts in the maize anther.Most cells of the maize subepidermal endothecium have starch-containing chloroplasts that do not conduct measurable photosynthesis in vitro.The maize anther contains chloroplasts in most subepidermal, endothecial cells. Although maize anthers receive sufficient light to photosynthesize in vivo and the maize anther transcribes >96% of photosynthesis-associated genes found in the maize leaf, no photosynthetic light reaction activity was detected in vitro. The endothecial cell layer should no longer be defined as a complete circle viewed transversely in anther lobes, because chloroplasts are observed only in cells directly beneath the epidermis and not those adjacent to the connective tissue. We propose that chloroplasts be a defining characteristic of differentiated endothecial cells and that nonsubepidermal endothecial cells that lack chloroplasts be defined as a separate cell type, the interendothecium.

Abstract

The biotrophic smut fungus Ustilago maydis infects all aerial organs of maize (Zea mays) and induces tumors in the plant tissues. U. maydis deploys many effector proteins to manipulate its host. Previously, deletion analysis demonstrated that several effectors have important functions in inducing tumor expansion specifically in maize leaves. Here, we present the functional characterization of the effector See1 (Seedling efficient effector1). See1 is required for the reactivation of plant DNA synthesis, which is crucial for tumor progression in leaf cells. By contrast, See1 does not affect tumor formation in immature tassel floral tissues, where maize cell proliferation occurs independent of fungal infection. See1 interacts with a maize homolog of SGT1 (Suppressor of G2 allele of skp1), a factor acting in cell cycle progression in yeast (Saccharomyces cerevisiae) and an important component of plant and human innate immunity. See1 interferes with the MAPK-triggered phosphorylation of maize SGT1 at a monocot-specific phosphorylation site. We propose that See1 interferes with SGT1 activity, resulting in both modulation of immune responses and reactivation of DNA synthesis in leaf cells. This identifies See1 as a fungal effector that directly and specifically contributes to the formation of leaf tumors in maize.

Abstract

With the exception of Ustilago maydis, smut fungi infecting monocotyledonous hosts systemically colonize infected plants and cause symptoms exclusively in the inflorescences. Ustilago may disinfects primordia of all aerial organs of maize (Zea mays L.) and results in the formation of large plant tumours. Previously, we have found that U. maydis infection of seedling leaves, adult leaves and tassels causes organ-specific transcriptional changes in both the pathogen and the host. Of particular interest, U. may disgenes encoding secreted proteins are differentially expressed depending on the colonized maize organ. Therefore, we hypothesized that the fungus secretes virulence-related proteins (effectors)that act in an organ-specific manner. Here, we present the identification and functional characterization of 20 presumptive organ-specific U. maydis effector genes. Ustilago maydis deletion strains for these genes were generated and tested for infectivity of maize seedling leaves and tassels. This approach identified 11 effector genes required for the full virulence of U. maydis. In nine cases, virulence was only affected in one of the tested plant organs. These results demonstrate that individual fungal effector proteins contribute to fungal virulence in an organ-specific manner.

Abstract

Compared to the diversity of other floral organs, the steps in anther ontogeny, final cell types, and overall organ shape are remarkably conserved among Angiosperms. Defects in pre-meiotic anthers that alter cellular composition or function typically result in male-sterility. Given the ease of identifying male-sterile mutants, dozens of genes with key roles in early anther development have been identified and cloned in model species, ordered by time of action and spatiotemporal expression, and used to propose explanatory models for critical steps in cell fate specification. Despite rapid progress, fundamental issues in anther development remain unresolved, and it is unclear if insights from one species can be applied to others. Here we construct a comparison of Arabidopsis, rice, and maize immature anthers to pinpoint distinctions in developmental pace. We analyze the mechanisms by which archesporial (pre-meiotic) cells are specified distinct from the soma, discuss what constitutes meiotic preparation, and review what is known about the secondary parietal layer and its terminal periclinal division that generates the tapetal and middle layers. Finally, roles for small RNAs are examined, focusing on the grass-specific phasiRNAs.

Abstract

Precise somatic and reproductive cell proliferation and differentiation in anthers are crucial for male fertility. Loss of function of the Male sterile 8 (Ms8) gene causes male sterility with multiple phenotypic defects first visible in the epidermal and tapetal cells. Here, we document the cloning of Ms8, which is a putative β-1,3-galactosyltransferase. Ms8 transcript is abundant in immature anthers with a peak at the meiotic stage; RNA expression is highly correlated with protein accumulation. Co-immunoprecipitation coupled with mass spectrometry sequencing identified several MS8-associated proteins, including arabinogalactan proteins, prohibitins, and porin. We discuss the hypotheses that arabinogalactan protein might be an MS8 substrate and that MS8 might be involved in maintenance of mitochondrial integrity.

Abstract

Male fertility in flowering plants relies on proper division and differentiation of cells in the anther, a process that gives rise to four somatic layers surrounding central germinal cells. The maize gene male sterility32 (ms32) encodes a basic helix-loop-helix (bHLH) transcription factor, which functions as an important regulator of both division and differentiation during anther development. After the four somatic cell layers are generated properly through successive periclinal divisions, in the ms32 mutant, tapetal precursor cells fail to differentiate, and, instead, undergo additional periclinal divisions to form extra layers of cells. These cells become vacuolated and expand, and lead to failure in pollen mother cell development. ms32 expression is specific to the pre-meiotic anthers and is distributed initially broadly in the four lobes, but as the anther develops, its expression becomes restricted to the innermost somatic layer, the tapetum. The ms32-ref mac1-1 double mutant is unable to form tapetal precursors and also exhibits excessive somatic proliferation leading to numerous, disorganized cell layers, suggesting a synergistic interaction between ms32 and mac1. Altogether, our results show that MS32 is a major regulator in maize anther development that promotes tapetum differentiation and inhibits periclinal division once a tapetal cell is specified.

Abstract

The basidiomycete Ustilago maydis is a ubiquitous pathogen of maize (Zea mays), one of the world's most important cereal crops. Infection by this smut fungus triggers tumor formation in aerial plant parts within which the fungus sporulates. Using confocal microscopy to track U. maydis infection on corn anthers through 7 dpi (days post-injection), we found that U. maydis is located on the epidermis on the first two days and by 3 dpi has reached all anther lobe cell types. Fungal infection can alter cell fate specification events, cell division patterns, host cell expansion, and host cell senescence depending on the developmental stage and cell type. Fungal impacts on tassel and plant growth were also quantified. Transcriptome profiling using a dual organism microarray identified thousands of anther genes affected by fungal infection 3 dpi during the cell fate specification and rapid cell proliferation phases of anther development. 4147 (17%) of anther-expressed genes were altered by infection, 2018 fungal genes were expressed in anthers, and 206 fungal secretome genes may be anther-specific. The results confirm that U. maydis deploys distinctive genes to cause disease in specific maize organs and begins to chart the mechanisms by which the host plant is manipulated to generate a tumor. This article is protected by copyright. All rights reserved.

Abstract

Proper regulation of anther differentiation is crucial for producing functional pollen, and defects in or absence of any anther cell type result in male sterility. To deepen understanding of processes required to establish premeiotic cell fate and differentiation of somatic support cell layers a cytological screen of maize male-sterile mutants has been conducted which yielded 42 new mutants including 22 mutants with premeiotic cytological defects (increasing this class fivefold), 7 mutants with postmeiotic defects, and 13 mutants with irregular meiosis. Allelism tests with known and new mutants confirmed new alleles of four premeiotic developmental mutants, including two novel alleles of msca1 and single new alleles of ms32, ms8, and ocl4, and two alleles of the postmeiotic ms45. An allelic pair of newly described mutants was found. Premeiotic mutants are now classified into four categories: anther identity defects, abnormal anther structure, locular wall defects and premature degradation of cell layers, and/or microsporocyte collapse. The range of mutant phenotypic classes is discussed in comparison with developmental genetic investigation of anther development in rice and Arabidopsis to highlight similarities and differences between grasses and eudicots and within the grasses.

Abstract

During maize anther development, somatic locular cells differentiate to support meiosis in the pollen mother cells. Meiosis is an important event during anther growth and is essential for plant fertility as pollen contains the haploid sperm. A subset of maize male sterile mutants exhibit meiotic failure, including ms8 (male sterile 8) in which meiocytes arrest as dyads and the locular somatic cells exhibit multiple defects. Systematic proteomic profiles were analysed in biological triplicates plus technical triplicates comparing ms8 anthers with fertile sibling samples at both the premeiotic and meiotic stages; proteins from 3.5 to 20 kDa were fractionated by 1-D PAGE, cleaved with Lys-C and then sequenced using a LTQ Orbitrap Velos MS paradigm. Three hundred and 59 proteins were identified with two or more assigned peptides in which each of those peptides were counted at least two or more times (0.4% peptide false discovery rate (FDR) and 0.2% protein FDR); 2761 proteins were identified with one or more assigned peptides (0.4% peptide FDR and 7.6% protein FDR). Stage-specific protein expression provides candidate stage markers for early anther development, and proteins specifically expressed in fertile compared to sterile anthers provide important clues about the regulation of meiosis. 49% of the proteins detected by this study are new to an independent whole anther proteome, and many small proteins missed by automated maize genome annotation were validated; these outcomes indicate the value of focusing on low molecular weight proteins. The roles of distinctive expressed proteins and methods for mass spectrometry of low molecular weight proteins are discussed.

Abstract

To ensure fertility, complex somatic and germinal cell proliferation and differentiation programs must be executed in flowers. Loss-of-function of the maize multiple archesporial cells 1 (mac1) gene increases the meiotically competent population and ablates specification of somatic wall layers in anthers. We report the cloning of mac1, which is the ortholog of rice TDL1A. Contrary to prior studies in rice and Arabidopsis in which mac1-like genes were inferred to act late to suppress trans-differentiation of somatic tapetal cells into meiocytes, we find that mac1 anthers contain excess archesporial (AR) cells that proliferate at least twofold more rapidly than normal prior to tapetal specification, suggesting that MAC1 regulates cell proliferation. mac1 transcript is abundant in immature anthers and roots. By immunolocalization, MAC1 protein accumulates preferentially in AR cells with a declining radial gradient that could result from diffusion. By transient expression in onion epidermis, we demonstrate experimentally that MAC1 is secreted, confirming that the predicted signal peptide domain in MAC1 leads to secretion. Insights from cytology and double-mutant studies with ameiotic1 and absence of first division1 mutants confirm that MAC1 does not affect meiotic cell fate; it also operates independently of an epidermal, Ocl4-dependent pathway that regulates proliferation of subepidermal cells. MAC1 both suppresses excess AR proliferation and is responsible for triggering periclinal division of subepidermal cells. We discuss how MAC1 can coordinate the temporal and spatial pattern of cell proliferation in maize anthers.

Abstract

Mu killer contains a partial inverted duplication of the mudrA transposase gene and two copies of the terminal inverted repeat A (TIRA) region of the master MuDR element of maize. Mu killer can effectively silence single copy MuDR/Mu lines, and it is proposed that a ∼4 kb hairpin RNA is generated by read through transcription from a flanking gene and that this transcript serves as a substrate for siRNA production. Mu killer was sequenced, except for a recalcitrant portion in the center of the locus, and shown to be co-linear with mudrA as originally proposed. The ability of the dominant Mu killer locus to silence a standard high copy number MuDR/Mu transposon line was evaluated. After two generations of exposure, about three quarters of individuals were silenced indicating reasonable effectiveness as measured by the absence of mudrA transposase transcripts. Mu killer individuals that effectively silenced MuDR expressed two short antisense transcripts. In contrast, Mu killer individuals that failed to silence MuDR expressed multiple sense transcripts, derived from read through transcription initiating in a flanking gene, but no antisense transcripts were detected.

Abstract

Maize male reproductive development is complex and lengthy, and anther formation and pollen maturation are precisely and spatiotemporally regulated. Here, we document that callose, somatic, and microspore defect 1 (csmd1), a new male-sterile mutant, has both pre-meiotic somatic and post-meiotic gametophyte and somatic defects. Chromosome behavior and cell developmental events were monitored by nuclear staining viewed by bright field microscopy; cell dimensions were charted by Volocity analysis of confocal microscopy images. Aniline blue staining and quantitative assays were performed to record callose deposition, and expression of three callose synthase genes was measured by qRT-PCR. Despite numerous defects and unlike other maize male-sterile mutants that show growth arrest coincident with locular defects, csmd1 anther elongation is nearly normal. Pre-meiotically and during prophase I, there is excess callose surrounding the meiocytes. Post-meiotically csmd1 epidermal cells have impaired elongation but excess longitudinal divisions, and uninucleate microspores cease growth; the microspore nucleoli degrade followed by cytoplasmic vacuolization and haploid cell collapse. The single vascular bundle within csmd1 anthers senesces precociously, coordinate with microspore death. Although csmd1 anther locules contain only epidermal and endothecial cells at maturity, locules are oval rather than collapsed, indicating that these two cell types suffice to maintain an open channel within each locule. Our data indicate that csmd1 encodes a crucial factor important for normal anther development in both somatic and haploid cells, that excess callose deposition does not cause meiotic arrest, and that developing pollen is not required for continued maize anther growth.

Abstract

A cold night can follow a hot day, and because they cannot move, plants subjected to such temperature fluctuations must acclimate on the basis mainly of pre-existing proteins. Zhang et al. report in a paper in BMC Plant Biology, however, that heat-induced cell death results from transcriptional activation of a kinase related to disease resistance factors and leading to a localized hypersensitive response. This specialized response reflects the failure of adaptations that normally enable plants to survive over a remarkable temperature range, by mechanisms that are not fully understood.

Abstract

UV-B radiation from normal solar fluence elicits physiological and developmental changes in plants under fluctuating environmental conditions. Most UV photobiology studies in plants utilize controlled greenhouse and growth chamber environments in which few conditions vary except the brief presence of UV-B radiation. Our purpose was to compare responses to UV-B in irradiated and shielded maize organs in field (natural solar plus 2x solar supplementation for defined periods) and greenhouse (2x solar supplementation only) conditions during a 4 hour exposure. Three parameters were assessed--transcripts, proteins, and metabolites--to determine the degree of overlap in maize responses in field and greenhouse conditions. We assessed irradiated leaves, and both shielded leaves and immature ears. After comparing transcriptome, proteome and metabolome profiles, we find there are more differences than similarities between field and greenhouse responses.

Abstract

Under normal solar fluence, UV-B damages macromolecules, but it also elicits physiological acclimation and developmental changes in plants. Excess UV-B decreases crop yield. Using a treatment twice solar fluence, we focus on discovering signals produced in UV-B-irradiated maize leaves that translate to systemic changes in shielded leaves and immature ears.Using transcriptome and proteomic profiling, we tracked the kinetics of transcript and protein alterations in exposed and shielded organs over 6 h. In parallel, metabolic profiling identified candidate signaling molecules based on rapid increase in irradiated leaves and increased levels in shielded organs; pathways associated with the synthesis, sequestration, or degradation of some of these potential signal molecules were UV-B-responsive. Exposure of just the top leaf substantially alters the transcriptomes of both irradiated and shielded organs, with greater changes as additional leaves are irradiated. Some phenylpropanoid pathway genes are expressed only in irradiated leaves, reflected in accumulation of pathway sunscreen molecules. Most protein changes detected occur quickly: approximately 92% of the proteins in leaves and 73% in immature ears changed after 4 h UV-B were altered by a 1 h UV-B treatment.There were significant transcriptome, proteomic, and metabolomic changes under all conditions studied in both shielded and irradiated organs. A dramatic decrease in transcript diversity in irradiated and shielded leaves occurs between 0 h and 1 h, demonstrating the susceptibility of plants to short term UV-B spikes as during ozone depletion. Immature maize ears are highly responsive to canopy leaf exposure to UV-B.

Abstract

Current software for storing and displaying records of genetic crosses does not provide an easy way to determine the lineage of an individual. The genetic records family tree (GRFT) applet processes records of genetic crosses and allows researchers to quickly visualize lineages using a family tree construct and to access other information from these records using any Internet browser. Users select from three display features: (1) a family tree view which displays a color-coded family tree for an individual, (2) a sequential list of crosses, and (3) a list of crosses matching user-defined search criteria. Each feature contains options to specify the number of records shown and the latter two contain an option to filter results by the owner of the cross. The family tree feature is interactive, displaying a popup box with genetic information when the user mouses over an individual and allowing the user to draw a new tree by clicking on any individual in the current tree. The applet is written in JavaScript and reads genetic records from a tab-delimited text file on the server, so it is cross-platform, can be accessed by anyone with an Internet connection, and supports almost instantaneous generation of new trees and table lists. Researchers can use the tool with their own genetic cross records for any sexually reproducing organism. No additional software is required and with only minor modifications to the script, researchers can add their own custom columns. GRFT's speed, versatility, and low overhead make it an effective and innovative visualization method for genetic records. A sample tool is available at http://stanford.edu/walbot/grft-sample.html.

Abstract

Spontaneous silencing of MuDR/Mu transposons occurs in approximately 10-100% of the progeny of an active plant, and once silenced reactivation is very rare. To date, only radiation treatments have reactivated silenced Mu; for example UV-B radiation reactivated Mutator activities. Here we have investigated possible mechanisms by which UV-B could reactivate Mu transposons by monitoring transcript abundance, epigenetic DNA marks, and chromatin factors associated with these elements. We demonstrate that both mudrA and B transcripts are expressed at higher levels after an 8 h-UV-B treatment, in both active Mutator and silencing plants, and that different chromatin remodeling events occur in the promoter regions of MuDR than in non-autonomous Mu1 elements. Increased transcript abundance is accompanied by an increase in histone H3 acetylation and by decreased DNA and H3K9me2 methylation. No changes in siRNA levels were detected. In contrast, the decrease in H3K9me2 present at Mu elements after UV-B is significant in silencing plants, suggesting that early changes in H3 methylation in K9, chromatin remodeling, and transcription factor binding contribute directly to transposon reactivation by UV-B in maize.

Abstract

Full-length cDNA (FLcDNA) sequencing establishes the precise primary structure of individual gene transcripts. From two libraries representing 27 B73 tissues and abiotic stress treatments, 27,455 high-quality FLcDNAs were sequenced. The average transcript length was 1.44 kb including 218 bases and 321 bases of 5' and 3' UTR, respectively, with 8.6% of the FLcDNAs encoding predicted proteins of fewer than 100 amino acids. Approximately 94% of the FLcDNAs were stringently mapped to the maize genome. Although nearly two-thirds of this genome is composed of transposable elements (TEs), only 5.6% of the FLcDNAs contained TE sequences in coding or UTR regions. Approximately 7.2% of the FLcDNAs are putative transcription factors, suggesting that rare transcripts are well-enriched in our FLcDNA set. Protein similarity searching identified 1,737 maize transcripts not present in rice, sorghum, Arabidopsis, or poplar annotated genes. A strict FLcDNA assembly generated 24,467 non-redundant sequences, of which 88% have non-maize protein matches. The FLcDNAs were also assembled with 41,759 FLcDNAs in GenBank from other projects, where semi-strict parameters were used to identify 13,368 potentially unique non-redundant sequences from this project. The libraries, ESTs, and FLcDNA sequences produced from this project are publicly available. The annotated EST and FLcDNA assemblies are available through the maize FLcDNA web resource (www.maizecdna.org).

Abstract

To address the role of small regulatory RNAs in rice development, we generated a large data set of small RNAs from mature leaves and developing roots, shoots, and inflorescences. Using a spatial clustering algorithm, we identified 36,780 genomic groups of small RNAs. Most consisted of 24-nt RNAs that are expressed in all four tissues and enriched in repeat regions of the genome; 1029 clusters were composed primarily of 21-nt small RNAs and, strikingly, 831 of these contained phased RNAs and were preferentially expressed in developing inflorescences. Thirty-eight of the 24-mer clusters were also phased and preferentially expressed in inflorescences. The phased 21-mer clusters derive from nonprotein coding, nonrepeat regions of the genome and are grouped together into superclusters containing 10-46 clusters. The majority of these 21-mer clusters (705/831) are flanked by a degenerate 22-nt motif that is offset by 12 nt from the main phase of the cluster. Small RNAs complementary to these flanking 22-nt motifs define a new miRNA family, which is conserved in maize and expressed in developing reproductive tissues in both plants. These results suggest that the biogenesis of phased inflorescence RNAs resembles that of tasiRNAs and raise the possibility that these novel small RNAs function in early reproductive development in rice and other monocots.

Abstract

Sensitive and reproducible genotyping tools are fundamental in interpreting and substantiating genetic data. In cases where alternative assays like PCR are not applicable, a sensitive genomic Southern protocol is needed. Our maize gene discovery work using the RescueMu transgenic lines was such a task. The direct proof of each new germinal insertion event can be assessed only on a genomic DNA hybridization analysis, and therefore we developed the following protocol to screen efficiently through hundreds up to thousands of samples in a relatively short time. The DNA extraction protocol was scaled to accommodate samples processed in a microcentrifuge with consistent yield of approximately 50 microg of high molecular weight DNA. A trained person can easily process several hundred samples in a few days. Once the DNA is extracted, final results can be obtained routinely within a week on approximately 100 or more samples, depending on the capacity of the electrophoresis and hybridization apparatus available. Under our optimized conditions, the method described below generates blots with high sensitivity and low background even after repeated stripping and reprobing. Single to low-copy transgenes as well as maize genomic sequences can be detected consistently. The nonradioactive DNA probes employed are not only safer, compared to the conventional radioactive probes, but also greatly shorten the exposure time. Confident estimation of copy number - as good as quantitative PCR - and visualization of transgene complexity are just a few more advantages of this protocol.

Abstract

The transgenic RescueMu lines were designed for and successfully used in our maize gene discovery project. The pBluescript-containing RescueMu transposon can be readily recovered by a procedure called plasmid rescue. Plasmid rescue is a technique for recovering bacterial plasmids from transgenic eukaryotic genomic DNA. Total maize DNA was first digested with restriction enzyme(s), ligated, and then transformed into E. coli cells. Colonies were recovered under selection against the antibiotic marker(s) in the transgene vector. Ampicillin or carbenicillin was used for RescueMu transgene recovery. The flanking genomic sequences at RescueMu insertion sites were simultaneously captured and then sequenced using RescueMu-readout primers. Genomic DNA from an individual plant or from pooled samples of up to approximately 50 plants could be used in a single rescue. Because the majority of transgenic constructs currently used in flowering plants were made in the form of plasmids, this protocol could therefore be adapted by and useful to researchers involved in other transgenic work and be versatile for characterizing transgene loci.

Abstract

Maize seedling transcriptome responses to six abiotic perturbations (heat, cold, darkness, desiccation, salt, ultraviolet-B) were analysed. Approximately 7800 transcripts were expressed in one or more treatments compared with light-grown seedlings plus juvenile leaves from field-grown plants. Approximately 5200 transcripts were expressed in one or more treatments and absent in light-grown seedlings. Approximately 2000 transcripts were unique to one treatment. Salt and heat elicited the largest number of transcript changes; however, salt resulted in mostly a decreased abundance of transcripts, whereas heat shock resulted in mostly an increased abundance of transcripts. A total of 384 transcripts were common to all stress treatments and not expressed in light-grown seedlings; 146 transcripts were present in light-grown seedlings and absent from all stress treatments. A complex pattern of overlapping transcripts between treatments was found, and a significant pattern of congruence in the direction of transcript change between pairs of treatments was uncovered. From the analysis, it appears that the scope of gene expression changes is determined by the challenge, indicating specificity in perception and response. Nonetheless, transcripts regulated by multiple responses are generally affected in the same manner, indicating common or converging regulatory networks. The data are available for additional analysis through a searchable database.

Abstract

RNAi knockdown lines targeting two putative chromatin factors (a methyl-CpG-binding domain protein MBD101 and a chromatin remodeling complex protein CHC101) exhibit identical phenotypic consequences after UV-B exposure including necrosis in adult leaves and seedling death. Here we report that these RNAi lines exhibit substantially different transcriptome changes assessed on a 44 K Agilent oligonucleotide array platform compared to each other and to UV-B tolerant non-transgenic siblings both before and after 8 h of UV-B exposure. Adult maize leaves express approximately 26,000 transcript types under greenhouse growth conditions; after 8 h of UV-B exposure 267 transcripts exhibit an expression change in the B73 control line. Most of these transcript abundance changes in B73 after UV-B treatment are not found in the two RNAi knockdown lines: 119 upregulated transcript types and 128 downregulated types are uniquely modulated in B73. The mbd101 RNAi line shows many more line-specific transcript changes (897 up, 68 down) than either B73 or the chc101 line (72 up, 103 down). By functional analysis, the largest category of genes with predicted functions affected by UV-B is the DNA/chromatin binding group. Differential activation of suites of transcription factors in the control and transgenic lines are the likely explanation for the divergent transcriptome profiles.

Abstract

The nuclear proteomes of maize (Zea mays) lines that differ in UV-B tolerance were compared by two-dimensional gel electrophoresis after UV light treatment. Differential accumulation of chromatin proteins, particularly histones, constituted the largest class identified by mass spectrometry. UV-B-tolerant landraces and the B73 inbred line show twice as many protein changes as the UV-B-sensitive b, pl W23 inbred line and transgenic maize expressing RNA interference constructs directed against chromatin factors. Mass spectrometic analysis of posttranslational modifications on histone proteins demonstrates that UV-B-tolerant lines exhibit greater acetylation on N-terminal tails of histones H3 and H4 after irradiation. These acetylated histones are enriched in the promoter and transcribed regions of the two UV-B-upregulated genes examined; radiation-sensitive lines lack this enrichment. DNase I and micrococcal nuclease hypersensitivity assays indicate that chromatin adopts looser structures around the selected genes in the UV-B-tolerant samples. Chromatin immunoprecipitation experiments identified additional chromatin factor changes associated with the nfc102 test gene after UV-B treatment in radiation-tolerant lines. Chromatin remodeling is thus shown to be a key process in acclimation to UV-B, and lines deficient in this process are more sensitive to UV-B.

Abstract

During flowering, central anther cells switch from mitosis to meiosis, ultimately forming pollen containing haploid sperm. Four rings of surrounding somatic cells differentiate to support first meiosis and later pollen dispersal. Synchronous development of many anthers per tassel and within each anther facilitates dissection of carefully staged maize anthers for transcriptome profiling.Global gene expression profiles of 7 stages representing 29 days of anther development are analyzed using a 44 K oligonucleotide array querying approximately 80% of maize protein-coding genes. Mature haploid pollen containing just two cell types expresses 10,000 transcripts. Anthers contain 5 major cell types and express >24,000 transcript types: each anther stage expresses approximately 10,000 constitutive and approximately 10,000 or more transcripts restricted to one or a few stages. The lowest complexity is present during meiosis. Large suites of stage-specific and co-expressed genes are identified through Gene Ontology and clustering analyses as functional classes for pre-meiotic, meiotic, and post-meiotic anther development. MADS box and zinc finger transcription factors with constitutive and stage-limited expression are identified.We propose that the extensive gene expression of anther cells and pollen represents the key test of maize genome fitness, permitting strong selection against deleterious alleles in diploid anthers and haploid pollen. Because flowering plants show a substantial bias for male-sterile compared to female-sterile mutations, we propose that this fitness test is general. Because both somatic and germinal cells are transcriptionally quiescent during meiosis, we hypothesize that successful completion of meiosis is required to trigger maturation of anther somatic cells.

Abstract

Genetic gain in the yield of modern maize reflects increased stress tolerance. The manipulation of genes for deliberate alterations in tolerance relies on an understanding of the regulation and components of stress responses. Transcriptome analysis of an ultraviolet (UV) radiation time course with paired treatment and control measurements yielded groups of coordinately regulated genes and gene ontology processes. A comparison of the patterns of gene expression with patterns of morphological changes allowed the identification of physiologically relevant gene expression regulons. A set of genes significantly affected by UV radiation in maize leaves was selected by linear modelling plus order-restricted inference profile matches. This gene list was used to find upstream sequence motifs that predict the UV regulation of maize gene expression.

Abstract

Maize MuDR/Mu elements are one of the most active Class II transposons and are widely used for transposon tagging for gene cloning. The autonomous MuDR encodes a transposase, while diverse non-autonomous elements share similarity to MuDR only within their ~215 bp terminal inverted repeats (TIRs). Four independent Mu-induced mutable alleles of the anthocyanin pigment pathway Bronze2 (Bz2) locus have been sequenced; bz2-mu1, bz2-mu2, and bz2-mu3 contain Mu1 element insertions while bz2-mu4 contains a MuDR insertion. Somatic excision activity can be monitored for each allele as a purple spotted phenotype on the otherwise beige epidermal layer of the kernel. To study epigenetic silencing of Mu elements, we investigated inheritance of somatic transposition of these four reporter alleles, and using samples from leaves just below the ear and the tassel, DNA methylation status and mudrA expression were quantified through three consecutive generations. Percentages of spotted kernels when crossed into bz2 tester as pollen parent were lower than those when crossed with bz2 tester as ear parent in all bz2-mu alleles. The propensity for silencing in kernels with different frequencies of spotting was investigated. In the inactive lines, both MuDR and Mu1 elements were de novo methylated. The transposition frequency was negatively correlated with the level of Mu methylation and positively correlated with the level of mudrA transcript. The most reliable indicator of incipient silencing was a decrease in mudrA transcript levels in the leaf below the tassel, and this transcriptional silencing could precede methylation of Mu elements.

Abstract

DNA arrays based on short oligonucleotide (< or = 25-mer) probes are being developed for many species, and are being applied to quantify transcript abundance variation in species with high genetic diversity. To define the parameters necessary to design short oligo arrays for maize (Zea mays L.), a species with particularly high nucleotide (single nucleotide polymorphism, SNP) and insertion-deletion (indel) polymorphism frequencies, we analysed gene expression estimates generated for four maize inbred lines using a custom Affymetrix DNA array, and identified biases associated with high levels of polymorphism between lines. Statistically significant interactions between probes and maize inbreds were detected, affecting five or more probes (out of 30 probes per transcript) in the majority of cases. SNPs and indels were identified by re-sequencing; they are the primary source of probe-by-line interactions, affecting probeset level estimates and reducing the power of detecting transcript level variation between maize inbreds. This analysis identified 36,196 probes in 5118 probesets containing markers that may be used for genotyping in natural and segregating populations for association gene analysis and genetic mapping.

Abstract

UV-B radiation causes diverse morphological and physiological responses in plants, but the underlying mechanisms governing these integrated responses are unknown. In this study, we systematically surveyed responses of maize leaves to UV-B radiation using DIGE 2D gels and identified selected proteins by mass spectrometry and immunodetection analysis. To identify changes in protein accumulation in response to UV-B radiation, a line (b, pl W23) deficient in flavonoid sunscreen compounds and hence similar to commercial corn was used. In addition, its proteome in natural UV-B conditions was compared with that of two maize landraces from high altitudes (Cacahuacintle and Confite Puneño) that have improved UV-B tolerance. Protein patterns in adult maize leaves (Zea mays) were documented after growth for 21 days in sunlight depleted of UV-B radiation or growth in sunlight including an 8-h UV-B supplementation during 1 day in the field. We found that there is a very high correlation between previously documented mRNA accumulation assessed by microarray hybridization and quantitative real time reverse transcription-PCR and protein expression after UV-B irradiation in leaves of W23. Multiple isoforms were confirmed for some proteins; at least one protein, pyruvate, phosphate dikinase, is regulated post-translationally by phosphorylation by UV-B exposure. Proteins differentially regulated by UV-B radiation in W23 with higher levels under similar UV-B conditions in high altitude plants were also identified. These could be genetically fixed traits conferring UV-B tolerance and offer clues to specific adaptations to living in high ambient UV-B conditions.

Crosslinking of ribosomal proteins to RNA in maize ribosomes by UV-B and its effects on translation(1[w])PLANT PHYSIOLOGYCasati, P., Walbot, V.2004; 136 (2): 3319-3332

Abstract

Ultraviolet-B (UV-B) photons can cause substantial cellular damage in biomolecules, as is well established for DNA. Because RNA has the same absorption spectrum for UV as DNA, we have investigated damage to this cellular constituent. In maize (Zea mays) leaves, UV-B radiation damages ribosomes by crosslinking cytosolic ribosomal proteins S14, L23a, and L32, and chloroplast ribosomal protein L29 to RNA. Ribosomal damage accumulated during a day of UV-B exposure correlated with a progressive decrease in new protein production; however, de novo synthesis of some ribosomal proteins is increased after 6 h of UV-B exposure. After 16 h without UV-B, damaged ribosomes were eliminated and translation was restored to normal levels. Ribosomal protein S6 and an S6 kinase are phosphorylated during UV-B exposure; these modifications are associated with selective translation of some ribosomal proteins after ribosome damage in mammalian fibroblast cells and may be an adaptation in maize. Neither photosynthesis nor pigment levels were affected significantly by UV-B, demonstrating that the treatment applied is not lethal and that maize leaf physiology readily recovers.

Abstract

Anthocyanin biosynthesis is one of the most thoroughly studied enzymatic pathways in biology, but little is known about the molecular mechanisms of its final stage: the transport of the anthocyanin pigment into the vacuole. We have identified a multidrug resistance-associated protein (MRP), ZmMrp3, that is required for this transport process in maize (Zea mays). ZmMrp3 expression is controlled by the regulators of anthocyanin biosynthesis and mirrors the expression of other anthocyanin structural genes. Localization of ZmMRP3 in vivo shows its presence in the tonoplast, the site at which anthocyanin transport occurs. Mutants generated using antisense constructs have a distinct pigmentation phenotype in the adult plant that results from a mislocalization of the pigment as well as significant reduction in anthocyanin content, with no alteration in the anthocyanin species produced. Surprisingly, mutant plants did not show a phenotype in the aleurone. This appears to reflect the presence of a second, highly homologous gene, ZmMrp4, that is also coregulated with the anthocyanin pathway but is expressed exclusively in aleurone tissue. This description of a plant MRP with a role in the transport of a known endogenous substrate provides a new model system for examining the biological and biochemical mechanisms involved in the MRP-mediated transport of plant secondary metabolites.

Abstract

Depletion of stratospheric ozone has raised terrestrial levels of ultraviolet-B radiation (UV-B), an environmental change linked to an increased risk of skin cancer and with potentially deleterious consequences for plants. To better understand the processes of UV-B acclimation that result in altered plant morphology and physiology, we investigated gene expression in different organs of maize at several UV-B fluence rates and exposure times.Microarray hybridization was used to assess UV-B responses in directly exposed maize organs and organs shielded by a plastic that absorbs UV-B. After 8 hours of high UV-B, the abundance of 347 transcripts was altered: 285 were increased significantly in at least one organ and 80 were downregulated. More transcript changes occurred in directly exposed than in shielded organs, and the levels of more transcripts were changed in adult compared to seedling tissues. The time course of transcript abundance changes indicated that the response kinetics to UV-B is very rapid, as some transcript levels were altered within 1 hour of exposure.Most of the UV-B regulated genes are organ-specific. Because shielded tissues, including roots, immature ears, and leaves, displayed altered transcriptome profiles after exposure of the plant to UV-B, some signal(s) must be transmitted from irradiated to shielded tissues. These results indicate that there are integrated responses to UV-B radiation above normal levels. As the same total UV-B irradiation dose applied at three intensities elicited different transcript profiles, the transcriptome changes exhibit threshold effects rather than a reciprocal dose-effect response. Transcriptome profiling highlights possible signaling pathways and molecules for future research.

Abstract

The maize MuDR/Mu transposable elements are highly aggressive, and their activities are held in check by host developmental and epigenetic mechanisms. The Mutator regulatory element, MuDR, produces both sense and antisense transcripts. We have investigated the impact of the presence of antisense transcripts on the abundance of the corresponding sense messages and on the regulation of Mutator activities. We report that internal deletions in MuDR arise frequently in somatic tissues; preferential loss of the 3' untranslated region of mudrA and/or mudrB containing the intergenic region is correlated with chimeric sense mudrA/antisense mudrB and sense mudrB/antisense mudrA transcripts. Heritable internal deletions are extremely frequent (>10(-2) per element), and the resulting defective MuDR elements also encode antisense transcripts. Expression of endogenous or additional transgene-encoded antisense transcripts neither decreases sense transcript levels nor inhibits Mutator excision activity over the three generations examined. We propose that antisense transcripts produced by MuDR deletions are not dominant-negative regulators of Mutator activities.

Abstract

The glutathione S-transferase encoded by Bronze2 performs the last genetically defined step in maize anthocyanin biosynthesis, being required for pigment sequestration into vacuoles. The Bz2 primary transcript contains a single intron; in maize leaves both spliced and unspliced Bz2 transcripts are usually present and are predicted to encode 26 and 14 kDa proteins, respectively. To increase understanding of the role and regulation of Bz2 transcript splicing, we examined Bz2 expression during development in transgenic maize plants expressing a 35S:Bz2 (35S:mycBz2i) gene and, by transient expression analysis, in Black Mexican Sweet maize protoplasts. We show here that the gene is expressed in diverse tissues that lack functional copies of one or both transcription factors regulating anthocyanin synthesis, that transcript levels are much higher when the R/B plus C1/Pl transcription factors are present, and that the splicing decision depends on local sequence context. The predicted 14 kDa protein was never detected indicating that unspliced transcripts are likely to be non-coding. The native 26 kDa BZ2 protein is loosely membrane-bound, but it was detectable only in tissues accumulating anthocyanin. Consequently, BZ2 accumulation appears to be limited by stringent post-transcriptional regulation.

Abstract

Microarray hybridization was used to assess acclimation responses to four UV regimes by near isogenic maize (Zea mays) lines varying in flavonoid content. We found that 355 of the 2,500 cDNAs tested were regulated by UV radiation in at least one genotype. Among these, 232 transcripts are assigned putative functions, whereas 123 encode unknown proteins. UV-B increased expression of stress response and ribosomal protein genes, whereas photosynthesis-associated genes were down-regulated; lines lacking UV-absorbing pigments had more dramatic responses than did lines with these pigments, confirming the shielding role of these compounds. Sunlight filtered to remove UV-B or UV-B plus UV-A resulted in significant expression changes in many genes not previously associated with UV responses. Some pathways regulated by UV radiation are shared with defense, salt, and oxidative stresses; however, UV-B radiation can activate additional pathways not shared with other stresses.

Abstract

Particle bombardment was used to elucidate the function of Flavonoid3, a late-acting anthocyanin gene of the ornamental plant, carnation ( Dianthus caryophyllus L.). The fl3 mutation conditions dilute anthocyanin coloration that closely resembles phenotypes produced by the anthocyanin mutants bz2 of maize and an9 of petunia. Bz2 and An9 encode glutathione S-transferases (GSTs) involved in vacuolar sequestration of anthocyanins. Constructs containing either of these or another late-function maize gene, Bronze1 (UDPglucose:flavonol 3- O-glucosyltransferase), were introduced via microprojectile bombardment into fl3 petals. Complementation resulted only from Bz2 and An9, indicating that Fl3 encodes a GST involved in the transport of anthocyanins to the vacuole. The observed result in carnation, an angiosperm phylogenetically distant from maize and petunia, indicates that GST activity might be a universal step in the anthocyanin pathway. Microprojectile bombardment was used to identify late-pathway anthocyanin mutations, which may be responsible for the pale anthocyanin coloration of important cultivars in many species but which can be difficult to characterize by other means.

Abstract

The Maize Gene Discovery Project (MGDP) is a 5-year NSF-funded plant genome initiative that began in 1998. The MGDP collaboration involves researchers at six universities from diverse disciplines with the common goal of discovering new maize genes and developing tools for the phenotypic characterization of maize mutants. The project utilizes several approaches: EST sequencing, cDNA microarray production, and the discovery of gene function and genomic sequence through the use of a recombinant Mu1 transposon ( RescueMu). Current achievements of the MGDP (NSF 98-72657) include the sequencing of over 120,000 maize ESTs from diverse cDNA libraries, and over 70,000 RescueMu flanking sequences, as well as the cataloguing of mutant seed and cob phenotypes of 23,000 maize ears, 6,200 families of maize seedlings, and 4,000 families of adult maize plants carrying MuDR/Mu and RescueMu insertion alleles. A consolidation of over 24,000 unique sequences from 19 libraries has been made into the first two of the planned set of four "Unigene" microarray slides. In addition, slides for four EST libraries have been produced. These microarray slides, EST clones, library plates of immortalized RescueMu bacterial cultures, and seed are all available online (http://www.zmdb.iastate.edu). The ZmDB website posts periodic assemblies of all maize EST and genomic sequences available from GenBank. ZmDB is also a portal for sequence analysis software designed to aid in gene discovery: MuSeqBox, GeneSeqer, and SplicePredictor. In addition, ZmDB contains links to other plant and genetics websites.

Abstract

Zea mays DataBase (ZmDB) seeks to provide a comprehensive view of maize (corn) genetics by linking genomic sequence data with gene expression analysis and phenotypes of mutant plants. ZmDB originated in 1999 as the Web portal for a large project of maize gene discovery, sequencing and phenotypic analysis using a transposon tagging strategy and expressed sequence tag (EST) sequencing. Recently, ZmDB has broadened its scope to include all public maize ESTs, genome survey sequences (GSSs), and protein sequences. More than 170 000 ESTs are currently clustered into approximately 20 000 contigs and about an equal number of apparent singlets. These clusters are continuously updated and annotated with respect to potential encoded protein products. More than 100 000 GSSs are similarly assembled and annotated by spliced alignment with EST and protein sequences. The ZmDB interface provides quick access to analytical tools for further sequence analysis. Every sequence record is linked to several display options and similarity search tools, including services for multiple sequence alignment, protein domain determination and spliced alignment. Furthermore, ZmDB provides web-based ordering of materials generated in the project, including ESTs, ordered collections of genomic sequences tagged with the RescueMu transposon and microarrays of amplified ESTs. ZmDB can be accessed at http://zmdb.iastate.edu/.

Abstract

MuDR controls transposition of the Mu transposable element family in Zea mays L. It produces two major transcripts: mudrA and mudrB, mudrA encodes the MURA transposase, but no specific function has been ascribed to mudrB, which lacks strong homology to known genes. Using transient expression assays in onion epidermal cells, we defined three monopartite nuclear localization signals (NLSs) of MURA; each was functionally sufficient for nuclear targeting of MURA:GUS fusion proteins. Interestingly, one NLS (NLS-A3) is produced by the splicing of the third intron. In contrast, there were no clear NLS in MURB, and the major form of MURB aggregated in the cytoplasm. Self-interaction of MURA and of MURB was also shown in a yeast two-hybrid assay. To test whether interactions of MURA and MURB can occur at the level of protein translocation into the nucleus, a cytoplasmically localized MURB:GFP was co-expressed with MURA or with the GUS fusion proteins. Co-expression did not change the localization pattern of either MURA or MURB; MURA and MURB do not detectably interact in a yeast two-hybrid assay. These results suggest that MURA and MURB do not mutually affect their localization, at least in the forms examined here.

Abstract

Assembly of 73,000 expressed sequence tags (ESTs) representing multiple organs and developmental stages of maize (Zea mays) identified approximately 22,000 tentative unique genes (TUGs) at the criterion of 95% identity. Based on sequence similarity, overlap between any two of nine libraries with more than 3,000 ESTs ranged from 4% to 20% of the constituent TUGs. The most abundant ESTs were recovered from only one or a minority of the libraries, and only 26 EST contigs had members from all nine EST sets (presumably representing ubiquitously expressed genes). For several examples, ESTs for different members of gene families were detected in distinct organs. To study this further, two types of micro-array slides were fabricated, one containing 5,534 ESTs from 10- to 14-d-old endosperm, and the other 4,844 ESTs from immature ear, estimated to represent about 2,800 and 2,500 unique genes, respectively. Each array type was hybridized with fluorescent cDNA targets prepared from endosperm and immature ear poly(A(+)) RNA. Although the 10- to 14-d-old postpollination endosperm TUGs showed only 12% overlap with immature ear TUGs, endosperm target hybridized with 94% of the ear TUGs, and ear target hybridized with 57% of the endosperm TUGs. Incomplete EST sampling of low-abundance transcripts contributes to an underestimate of shared gene expression profiles. Reassembly of ESTs at the criterion of 90% identity suggests how cross hybridization among gene family members can overestimate the overlap in genes expressed in micro-array hybridization experiments.

Abstract

A maize array was fabricated with 5,376 unique expressed sequence tag (EST) clones sequenced from 4-day-old roots, immature ears and adult organ cDNA libraries. To elucidate organ relationships, relative mRNA levels were quantified by hybridization with embryos, three maize vegetative organs (leaf blades, leaf sheaths and roots) from multiple developmental stages, husk leaves and two types of floral organs (immature ears and silks).Clustering analyses of the hybridization data suggest that maize utilizes both the PEPCK and NADP-ME C(4) photosynthetic routes as genes in these pathways are co-regulated. Husk RNA has a gene-expression profile more similar to floral organs than to vegetative leaves. Only 7% of the genes were highly organ specific, showing over a fourfold difference in at least one of 12 comparisons and 37% showed a two- to fourfold difference. The majority of genes were expressed in diverse organs with little difference in transcript levels. Cross-hybridization among closely related genes within multigene families could obscure tissue specificity. As a first step in elucidating individual gene-expression patterns, we show that 45-nucleotide oligo probes produce signal intensities and signal ratios comparable to PCR probes on the same matrix.Gene-expression profile studies with cDNA microarrays provide a new molecular tool for defining plant organs and their relationships and for discovering new biological processes in silico. cDNA microarrays are insufficient for differentiating recently duplicated genes. Gene-specific oligo probes printed along with cDNA probes can query individual gene-expression profiles and gene families simultaneously.

Abstract

The completed Arabidopsis genome seems to be of limited value as a model for maize genomics. In addition to the expansion of repetitive sequences in maize and the lack of genomic micro-colinearity, maize-specific or highly-diverged proteins contribute to a predicted maize proteome of about 50,000 proteins, twice the size of that of Arabidopsis.

Abstract

RescueMu, a Mu1 element containing a bacterial plasmid, is mobilized by MuDR in transgenic maize. Somatic excision from a cell-autonomous marker gene yields >90% single cell sectors; empty donor sites often have deletions and insertions, including up to 210 bp of RescueMu/Mu1 terminal DNA. Late somatic insertions are contemporaneous with excisions, suggesting that "cut-and-paste" transposition occurs in the soma. During reproduction, RescueMu transposes infrequently from the initial transgene array, but once transposed, RescueMu is suitable for high throughput gene mutation and cloning. As with MuDR/Mu elements, heritable RescueMu insertions are not associated with excisions. Both somatic and germinal RescueMu insertions occur preferentially into genes and gene-like sequences, but they exhibit weak target site preferences. New insights into Mu behaviors are discussed with reference to two models proposed to explain the alternative outcomes of somatic and germinal events: a switch from somatic cut-and-paste to germinal replicative transposition or to host-mediated gap repair from sister chromatids.

Abstract

The extent of imprinting at R-r, frequency of paramutation at B-Intense and Pl, and epigenetic silencing of Mu transposons were evaluated in the W23 and A188 inbred lines of maize. All types of epigenetic phenomena affecting these loci of the anthocyanin pathway occurred more frequently in the W23 inbred line. Absence of down-regulation was dominant in F1 hybrid progeny. Identical alleles programme lower anthocyanin accumulation in A188 than in W23, and A188 plants develop more rapidly than W23. The possibilities that specific genetic factors, intrinsic gene expression levels and/or the rapidity of the life cycle modulate epigenetic gene controls are discussed.

Abstract

Since the structure of the DNA molecule was identified half a century ago, the complete genome sequence has been determined for 37 prokaryotes and several eukaryotes. With the exponential growth of genetic information, bioinformatics has attempted to predict gene locations and functions in cyberspace prior to experimental confirmation at the bench.

Abstract

Transgenic maize expressing luciferase under the control of the mudrB terminal inverted repeat promoter (TIRB) of the MuDR transposon was assayed for transgene expression in active and inactive Mutator lines. We find that active MuDR elements increase TIRB-luciferase expression by 2- to 10-fold, relative to nonMuDR or silenced MuDR lines, in embryonic leaves in 75% of plants tested. However, this increase does not persist in juvenile and adult leaves. In pollen, TIRB-luciferase expression is up to 100-fold higher than in leaves but is unaffected by the presence or absence of active MuDR. Because the MuRA transposase binds to a motif within TIRB, we hypothesize that MURA may act as a weak transcriptional activator of TIRB or may partly inhibit host-induced silencing of TIRB in active Mutator lines during the early stages of somatic growth. Our results contrast with those for the maize transposon Spm, in which the TNPA transposase acts as a repressor of the Spm promoter in active Spm lines.

Abstract

The Mu transposons of maize are under stringent developmental control. Elements excise at high frequencies in terminally dividing somatic cells, but not in meristems. Mu elements in germinal cells amplify, without excision, and insert throughout the genome. All activities require MuDR, which encodes two genes, mudrA and mudrB, whose near-identical promoters are located in the transposon terminal inverted repeats (TIR). We have fused the 216 bp TIR of the mudrB gene to GUS and luciferase reporters. We demonstrate that TIRB programs reporter expression in diverse, meristematic somatic cells, paradoxically in those cells in which Mu excisions are repressed. In germinal cells, immature tassel and mature pollen, reporter expression increases up to 20-fold compared to leaf. By RNA blot hybridization, we demonstrate that endogenous mudrB and mudrA transcripts increase significantly in mature pollen; sequence comparisons demonstrate that the MuDR TIRs contain plant cell-cycle enhancer motifs and functionally defined pollen enhancers. Therefore, the MuDR TIR promoters are developmentally regulated in both somatic and germinal tissues. Because database sequence analysis suggests that the MuDR TIR enhancers should be functional in both monocots and dicots, we suggest that the native MuDR promoter be used in attempts to transfer the unique behavior of Mu transposition to heterologous hosts.

Abstract

AN9 is a glutathione S-transferase from petunia (Petunia hybrida) required for efficient anthocyanin export from the site of synthesis in the cytoplasm into permanent storage in the vacuole. For many xenobiotics it is well established that a covalent glutathione (GSH) tag mediates recognition of molecules destined for vacuolar sequestration by a tonoplast-localized ATP-binding cassette pump. Here we inquired whether AN9 catalyzes the formation of GSH conjugates with flavonoid substrates. Using high-performance liquid chromatography analysis of reaction mixtures containing enzyme, GSH, and flavonoids, including anthocyanins, we could detect neither conjugates nor a decrease in the free thiol concentration. These results suggest that no conjugate is formed in vitro. However, AN9 was shown to bind flavonoids using three assays: inhibition of the glutathione S-transferase activity of AN9 toward the common substrate 1-chloro 2,4-dinitrobenzene, equilibrium dialysis, and tryptophan quenching. We conclude that AN9 is a flavonoid-binding protein, and propose that in vivo it serves as a cytoplasmic flavonoid carrier protein.

Abstract

Transposon mutagenesis facilitates gene discovery by tagging genes for cloning. New genomics projects are now cataloging transposon insertion sites to define all maize genes. Once identified, transposon insertions are 'hot spots' for generating new alleles that are useful in functional studies.

Abstract

The MuDR element responsible for Mutator activities in maize encodes two genes, mudrA and mudrB. Each encodes multiple transcripts hypothesized to regulate, directly or indirectly, the unique late timing and switch in transposition mechanism during maize development. mudrA, which encodes the MURA transposase, is unstable in bacterial plasmids, a technical problem solved by using phage M13 as a vector to prepare DNA for biolistic transformation. In transgenic maize, a single 2.7-kb mudrA cDNA predicted to encode an 823-amino acid protein is sufficient to catalyze late somatic excisions, despite removal of the native promoter, alternative transcription start sites, known introns, polymorphic 5' and 3' untranslated sequences, and the mudrB gene. These results suggest that post-translational regulation confers Mu excision timing. The transgene is active in lines containing silencing MuDR elements. This suggests that endogenous MuDR transposons do not measurably immunize the host against expression of a homologous transgene.

Abstract

Zea mays DataBase (ZmDB) is a repository and analysis tool for sequence, expression and phenotype data of the major crop plant maize. The data accessible in ZmDB are mostly generated in a large collaborative project of maize gene discovery, sequencing and phenotypic analysis using a transposon tagging strategy and expressed sequence tag (EST) sequencing. ESTs constitute most of the current content. Database search tools, convenient links to external databases, and novel sequence analysis programs for spliced alignment are provided and together serve as an efficient protocol for gene discovery by sequence inspection. ZmDB can be accessed at http://zmdb. iastate.edu. ZmDB also provides web-based ordering of materials generated in the project, including EST and genomic DNA clones, seeds of mutant plants and microarrays of amplified EST and genomic DNA sequences.

Abstract

Previous studies have established that splice site selection and splicing efficiency in plants depend strongly on local compositional contrast consisting of high exon G+C content relative to high intron U content. The combinatorial model of plant intron recognition posits that splice site sequences as well as local intron and exon sequences contribute to splice site selection and splicing efficiency. Most of the previous studies used synthetic or chimeric constructs, often tested in heterologous hosts. To perform a more critical test of the combinatorial model in a native context, the single intron of the maize Bronze2 gene and its flanking exons were modified by site-directed mutagenesis. Splicing efficiency was tested in maize protoplasts. Results show that a higher U content in the flanking 5' exon, whether close to or distant from the 5' splice site, did not modify splicing efficiency. Decreasing exon G+C content dramatically impaired splicing. Increasing intron G+C content or decreasing intron U content adversely impacted splicing. In all constructs splicing occurred exclusively at the original 5' and 3' splice sites. These results are consistent with the hypothesis that exon G+C content and intron U content contribute separate but complementary aspects of intron definition in the native Bz2 transcript.

Abstract

Glutathione S-transferases (GSTs) traditionally have been studied in plants and other organisms for their ability to detoxify chemically diverse herbicides and other toxic organic compounds. Anthocyanins are among the few endogenous substrates of plant GSTs that have been identified. The Bronze2 (Bz2) gene encodes a type III GST and performs the last genetically defined step of the maize anthocyanin pigment pathway. This step is the conjugation of glutathione to cyanidin 3-glucoside (C3G). Glutathionated C3G is transported to the vacuole via a tonoplast Mg-ATP-requiring glutathione pump (GS-X pump). Genetically, the comparable step in the petunia anthocyanin pathway is controlled by the Anthocyanin9 (An9) gene. An9 was cloned by transposon tagging and found to encode a type I plant GST. Bz2 and An9 have evolved independently from distinct types of GSTs, but each is regulated by the conserved transcriptional activators of the anthocyanin pathway. Here, a phylogenetic analysis is presented, with special consideration given to the origin of these genes and their relaxed substrate requirements. In particle bombardment tests, An9 and Bz2 functionally complement both mutants. Among several other GSTs tested, only soybean GmGST26A (previously called GmHsp26A and GH2/4) and maize GSTIII were found to confer vacuolar sequestration of anthocyanin. Previously, these genes had not been associated with the anthocyanin pathway. Requirements for An9 and Bz2 gene function were investigated by sequencing functional and nonfunctional germinal revertants of an9-T3529, bz2::Ds, and bz2::Mu1.

Abstract

To date, mobile Mu transposons and their autonomous regulator MuDR have been found only in the two known Mutator lines of maize and their immediate descendants. To gain insight into the origin, organization, and regulation of Mutator elements, we surveyed exotic maize and related species for cross-hybridization to MuDR. Some accessions of the mexican land race Zapalote chico contain one to several copies of full-length, unmethylated, and transcriptionally active MuDR-like elements plus non-autonomous Mu elements. The sequenced 5.0-kb MuDR-Zc element is 94.6% identical to MuDR, with only 20 amino acid changes in the 93-kD predicted protein encoded by mudrA and ten amino acid changes in the 23-kD predicted protein of mudrB. The terminal inverted repeat (TIR) A of MuDR-Zc is identical to standard MuDR; TIRB is 11.2% divergent from TIRA. In Zapalote chico, mudrA transcripts are very rare, while mudrB transcripts are as abundant as in Mutator lines with a few copies of MuDR. Zapalote chico lines with MuDR-like elements can trans-activate reporter alleles in inactive Mutator backgrounds; they match the characteristic increased forward mutation frequency of standard Mutator lines, but only after outcrossing to another line. Zapalote chico accessions that lack MuDR-like elements and the single copy MuDR a1-mum2 line produce few mutations. New mutants recovered from Zapalote chico are somatically stable.

Abstract

Using a large set of plant gene sequences we compared individual introns to their flanking exons. Both Zea mays and Arabidopsis thaliana introns are U-rich but display no apparent bias for A. We identified fifteen 11-mer U-rich motifs as frequent elements of maize introns, and these are virtually absent from exons. By mutagenesis, we show that the single U-rich motif in the Bronze2 intron of maize plays a key role in intron processing in vivo.

Abstract

Heterologous introns are often inaccurately or inefficiently processed in higher plants. The precise features that distinguish the process of pre-mRNA splicing in plants from splicing in yeast and mammals are unclear. One contributing factor is the prominent base compositional contrast between U-rich plant introns and flanking G + C-rich exons. Inclusion of this contrast factor in recently developed statistical methods for splice site prediction from sequence inspection significantly improved prediction accuracy. We applied the prediction tools to re-analyze experimental data on splice site selection and splicing efficiency for native and more than 170 mutated plant introns. In almost all cases, the experimentally determined preferred sites correspond to the highest scoring sites predicted by the model. In native genes, about 90% of splice sites are the locally highest scoring sites within the bounds of the flanking exon and intron. We propose that, in most cases, local context (about 50 bases upstream and downstream from a potential intron end) is sufficient to account for intrinsic splice site strength, and that competition for transacting factors determines splice site selection in vivo. We suggest that computer-aided splice site prediction can be a powerful tool for experimental design and interpretation.

Abstract

The autonomous MuDR element of the Mutator (Mu) transposable element family of maize encodes at least two proteins, MURA and MURB. Based on amino acid sequence similarity, previous studies have reported that MURA is likely to be a transposase. The functional characterization of MURA has been hindered by the instability of its cDNA, mudrA, in Escherichia coli. In this study, we report the first successful stabilization and expression of MURA in Saccharomyces cerevisiae. Gel mobility shift assays demonstrate that MURA is a DNA-binding protein that specifically binds to sequences within the highly conserved Mu element terminal inverted repeats (TIRs). DNase I and 1,10-phenanthroline-copper footprinting of MURA-Mu1 TIR complexes indicate that MURA binds to a conserved approximately 32-bp region in the TIR of Mu1. In addition, MURA can bind to the same region in the TIRs of all tested actively transposing Mu elements but binds poorly to the diverged Mu TIRs of inactive elements. Previous studies have reported a correlation between Mu transposon inactivation and methylation of the Mu element TIRs. Gel mobility shift assays demonstrate that MURA can interact differentially with unmethylated, hemimethylated, and homomethylated TIR substrates. The significance of MURA's interaction with the TIRs of Mu elements is discussed in the context of what is known about the regulation and mechanisms of Mutator activities in maize.

Abstract

Plant introns are typically AU-rich or U-rich, and this feature has been shown to be important for splicing. In maize, however, about 20% of the introns exceed 50% GC, and most of them are efficiently spliced. A series of constructs has been designed to analyze the cis requirements for splicing of the GC-rich Bz2 maize intron and two other GC-rich intron derivatives. By manipulating exon, intron and splice site sequences it is shown that exons can play an important role in intron definition: changes in exon sequences can increase splicing efficiency of a GC-rich intron from 17% to 86%. The relative difference, or base compositional contrast, in GC and U content between exon and intron sequences in the vicinity of splice sites, rather than the absolute base-content of the intron or exons, correlates with splicing efficiency. It is also shown that GC-rich intron constructs that are poorly spliced can be partially rescued by an improved 3' splice site.

Abstract

We have studied the uptake of [3H]-medicarpin and its glutathione conjugate(s) into vacuolar membrane vesicles from etiolated hypocotyls of mung bean (Vigna radiata). Unconjugated medicarpin is taken up at a low rate in the presence or absence of MgATP. However, [3H]-medicarpin-glutathione conjugate(s), prepared by incubation of medicarpin with a total maize glutathione S-transferase preparation, is taken up more than four-fold faster than medicarpin in the presence of MgATP, and this uptake is MgATP-dependent. Uptake of medicarpin-glutathione was not significantly inhibited by the ionophore gramicidin-D, but was strongly inhibited by vanadate and the alternative transport substrate S-(2,4-dinitrophenyl) glutathione. Our results demonstrate, in a model system, the potential utilization of the high affinity, high capacity, uncoupler-insensitive glutathione conjugate pump for the vacuolar transport of an isoflavonoid phytoalexin.

Abstract

Photolyases are DNA repair enzymes that use energy from blue light to repair pyrimidine dimers. We report the isolation of an Arabidopsis thaliana mutant (uvr2-1) that is defective in photorepair of cyclobutylpyrimidine dimers (CPDs). Whereas uvr2-1 is indistinguishable from wild type in the absence of UV light, low UV-B levels inhibit growth and cause leaf necrosis. uvr2-1 is more sensitive to UV-B than wild type when placed under white light after UV-B treatment. In contrast, recovery in darkness or in light lacking photoreactivating blue light results in equal injury in uvr2-1 and wild type. The uvr2-1 mutant is unable to remove CPDs in vivo, and plant extracts lack detectable photolyase activity. This recessive mutation segregates as a single gene located near the top of chromosome 1, and is a structural gene mutation in the type II CPD photolyase PHR1. This mutant provides evidence that CPD photolyase is required for plant survival in the presence of UV-B light.

Abstract

The Bronze2 (Bz2) gene in maize (Zea mays) encodes a glutathione S-transferase that performs the last genetically defined step in anthocyanin biosynthesis--tagging anthocyanin precursors with glutathione, allowing for recognition and entry of anthocyanins into the vacuole. Here we show that Bz2 gene expression is highly induced by heavy metals such as cadmium. Treatment of maize seedlings with cadmium results in a 20-fold increase in Bz2 message accumulation and a 50-fold increase in the presence of the unspliced, intron-containing transcript. The increase in message levels during cadmium stress appears to result, at least in part, from activation of an alternative mRNA start site approximately 200 nucleotides upstream of the normal start site; this site is not used in unstressed or heat-stressed tissues. The effect of cadmium on the RNA splicing of Bz2 seems to be specific: splicing of other intron-containing maize genes, including a maize actin gene under the control of the cadmium-inducible Bz2 promoter, is unaffected by cadmium stress. Conversely, Bz2 intron splicing is not affected by other stress conditions that induce Bz2 gene expression, such as abscisic acid, auxin, or cold stress. Surprisingly, the increase in Bz2 mRNA during cadmium stress does not result in an increase in Bz2 glutathione S-transferase activity. We propose that an alternative protein may be encoded by Bz2 that has a role during responses to heavy metals.

Abstract

Activity of the Mutator transposons of Zea mays varies in different tissues and at different stages of development. In the soma, Mu elements excise at a high frequency late in tissue development. In germ cells, Mu elements rarely excise, but they amplify and insert at high levels around the time of meiosis. At all other times, Mu elements can duplicate and insert at a low frequency. To determine whether the patterns of Mutator activity correlate with tissue or cell-specific transcription of the regulatory transposon MuDR, we used in situ hybridization to localize the sense MuDR transcripts, mudrA and mudrB, in pistillate florets and embryos of four different maize Mutator stocks. We found mudrA and mudrB transcripts uniformly distributed in all tissues of immature, meristem-rich florets and in both somatic and germinal tissues of mature florets. In mature flowers, transcripts of both genes accumulate to high levels in the tapetal (endothelium) layer surrounding the embryo sac. We also found transcripts from the antisense strand of the mudrA gene in all cell types in the florets. In developing embryos, all MuDR transcripts were present in all tissues. Different Mutator stocks had characteristic accumulation patterns that were maintained throughout embryo development.

Abstract

The maize Bronze2 (Bz2) gene encodes a glutathione S-transferase that is required for anthocyanin pigment accumulation. Two classes of regulatory proteins, R and C1, are required for transcriptional activation of Bz2 and several additional structural genes. Functional domains of the Bz2 promoter were identified using Bz2 promoter-driven luciferase reporter genes electroporated into maize protoplasts together with R and C1 expression plasmids. Complete regulation was conferred by 224 nt of the Bz2 promoter. Within this region at least two separable regions are independently capable of conferring regulation by R and C1. Predicted regulatory elements corresponding to two classes of sequence motifs, the Myb-box homologous 'C1-motif', TAACTG/CAGTTA, and the G-box and E-box homologous 'R-motif', CACGTG, were shown to be important for full R and C1 activation of the Bz2 promoter. Expression of reconstructed Bz2 genes with mutated promoters was quantified using RNase protection, and this analysis confirmed results obtained using reporter genes.

Abstract

The MuDR element controls the transposition of the Mutator transposable element family in maize. Previous studies reported the presence of two major MuDR-homologous transcripts that correlate with Mutator activity. In this study, we describe the structure and processing of these two major transcripts. The transcripts are convergent, initiating from opposite ends of the element within the 220-bp terminal inverted repeats. The convergent transcripts do not overlap, and only 200 bp of internal MuDR sequences are not transcribed. Cloning and sequencing of multiple MuDR cDNAs revealed unusual intron/exon junctions, differential splicing, and multiple polyadenylation sites. RNase protection experiments indicated that some splicing failure occurs in young seedlings, and that a low level of antisense RNA exists for both transcripts. On a whole plant level, the presence of the major MuDR transcripts strictly correlates with Mutator activity in that no MuDR transcripts are observed in non-Mutator or inactive Mutator stocks. Examination of various tissues from active Mutator stocks indicates that the two transcripts are present in all organs and tissues tested, including those with no apparent transposition activity. This suggests that Mutator activity is not simply controlled by the level of the major MuDR transcripts.

Abstract

Glutathione S-transferases (GSTs) are enzymes that detoxify heterocyclic compounds (xenobiotics) by covalently linking glutathione to the substrate, forming a glutathione S-conjugate. A glutathione pump in the vacuolar membrane of barley actively sequesters herbicide-glutathione S-conjugates; glutathionation allows recognition and entry of the conjugates into vacuoles. The protein encoded by the Bronze-2 gene in maize performs the last genetically defined step in anthocyanin biosynthesis, resulting in the deposition of red and purple pigments in the vacuoles of maize tissues. We show here that Bz2 encodes a GST with activity in maize, transformed Arabidopsis thaliana plants and Escherichia coli. We demonstrate that anthocyanins extracted from maize protoplasts expressing BZ2 are conjugated with glutathione, and that vanadate, a known inhibitor of the glutathione pump in plant vacuolar membranes, inhibits the accumulation of anthocyanins in the vacuole. These results provide a biochemical function for BZ2, and suggest a common mechanism for the ability of plants to sequester structurally similar but functionally diverse molecules in the vacuole.

Abstract

Pulsed-field gel electrophoresis (PFGE) in combination with infrequently cutting restriction enzymes was used to investigate the structure of the mitochondrial (mt) genome of the maize variety Black Mexican Sweet (BMS). The mt genome of this variety was found to resemble that of the closely related B37N variety, with one recombination and five insertion/deletion events being sufficient to account for the differences observed between the two genomes. The majority of the BMS genome is organized as a number of subgenomic chromosomes with circular restriction maps. Several large repeated sequences are found in the BMS mt genome, but not all appear to be in recombinational equilibrium. No molecules large enough to contain the entire mt genome were discernible using these techniques. The mapping approach described here provides a means of quickly analyzing the large and complex mt genomes of plants.

Abstract

The mechanisms of intron recognition and processing have been well-studied in mammals and yeast, but in plants the biochemistry of splicing is not known and the rules for intron recognition are not clearly defined. To increase understanding of intron processing in plants, we have constructed new pairs of vectors, pSuccess and pFail, to assess the efficiency of splicing in maize cells. In the pFail series we use translation of pre-mRNA to monitor the amount of unspliced RNA. We inserted an ATG codon in the Bz2 (Bronze-2) intron in frame with luciferase: this construct will express luciferase activity only when splicing fails. In the pSuccess series the spliced message is monitored by inserting an ATG upstream of the Bz2 intron in frame with luciferase: this construct will express luciferase activity only when splicing succeeds. We show here, using both the wild-type Bz2 intron and the same intron with splice site mutations, that the efficiency of splicing can be estimated by the ratio between the luciferase activities of the vector pairs. We also show that mutations in the unique U-rich motif inside the intron can modulate splicing. In addition, a GC-rich insertion in the first exon increases the efficiency of splicing, suggesting that exons also play an important role in intron recognition and/or processing.

Abstract

The control of cell fate was investigated in the root epidermis of Arabidopsis thaliana. Two distinct types of differentiated epidermal cells are normally present: root-hair-bearing cells and hairless cells. In wild-type Arabidopsis roots, epidermal cell fate was found to be correlated with cell position, with root-hair cells located over radial walls between cortical cells, and with hairless cells located directly over cortical cells. This normal positional relationship was absent in ttg (transparent testa glabrous) mutants (lacking trichomes, anthocyanins, and seed coat mucilage); epidermal cells in all positions differentiate into root-hair cells. The opposite condition was generated in roots of transgenic Arabidopsis expressing the maize R (R-Lc) gene product (a putative TTG homologue) under the control of a strong promoter (CaMV35S), which produced hairless epidermal cells in all positions. In both the ttg and R-expressing roots, epidermal cell differentiation was affected at an early stage, prior to the onset of cell elongation or root-hair formation. The ttg mutations were also associated with abnormalities in the morphology and organization of cells within and surrounding the root apical meristem. The results indicate that alterations in TTG activity cause developing epidermal cells to misinterpret their position and differentiate into inappropriate cell types. This suggests that, in wild-type roots, TTG provides, or responds to, positional signals to cause differentiating epidermal cells that lie over cortical cells to adopt a hairless cell fate.

Abstract

The Arabidopsis mutant ttg lacks both trichomes (epidermal hairs) and anthocyanin pigments. Trichomes and anthocyanins are restored by the constitutive expression of the maize transcriptional regulator (R). The expression of an R-glucocorticoid receptor chimeric protein results in a steroid hormone-dependent, conditional allele of R that functions in whole Arabidopsis plants. The response of the chimeric protein to pulses of hormone was used to define the pattern and timing of trichome formation on the developing leaf epidermis. Each adaxial epidermal leaf cell appears to have an equal probability of differentiating into a trichome; there is a temporal zone of decision for trichome cell fate that proceeds as a wave from the tip to the base of developing leaves.

Abstract

The Mutator transposable element system of maize is the most active transposable element system characterized in higher plants. While Mutator has been used to generate and tag thousands of new maize mutants, the mechanism and regulation of its transposition are poorly understood. The Mutator autonomous element, MuDR, encodes two proteins: MURA and MURB. We have detected an amino acid sequence motif shared by MURA and the putative transposases of a group of bacterial insertion sequences. Based on this similarity we believe that MURA is the transposase of the Mutator system. In addition we have detected two rice cDNAs in genbank with extensive similarity to MURA. This sequence similarity suggests that a Mutator-like element is present in rice. We believe that Mutator, a group of bacterial insertion sequences, and an uncharacterized rice transposon represent members of a family of transposable elements.

Abstract

Diverse flavonoid compounds are widely distributed in angiosperm families. Flavonoids absorb radiation in the ultraviolet (UV) region of the spectrum, and it has been proposed that these compounds function as UV filters. We demonstrate that the DNA in Zea mays plants that contain flavonoids (primarily anthocyanins) is protected from the induction of damage caused by UV radiation relative to the DNA in plants that are genetically deficient in these compounds. DNA damage was measured with a sensitive and simple assay using individual monoclonal antibodies, one specific for cyclobutane pyrimidine dimer damage and the other specific for pyrimidine(6,4)pyrimidone damage.

Abstract

Intron recognition in Angiosperms is hypothesized to require AU-rich motifs within introns. In this report we examined the role of AU-rich motifs in pre-mRNA processing. AU-rich segments of maize introns inserted near the single intron of the maize Bronze-2(Bz2) gene result in alternative splicing. Other insertions of AU-rich sequence in the Bz2 cDNA resulted in de novo intron creation using splice junctions at the edges of the AU-rich region. Surprisingly, the five AU-rich inserts that we tested also caused polyadenylation, even though none had been selected for that function in plants. Insertions of GC-rich sequence into Bz2 did not cause either splicing or polyadenylation. We propose that AU-rich motifs are a general signal for RNA processing in maize and that in the absence of a 5' splice site, polyadenylation is the default pathway.

Abstract

Plant introns are generally short (< 200 nt) and AU-rich, and an elevated AU content is necessary for efficient splicing. Further, an intron in some plant genes enhances gene expression by a post-transcriptional mechanism that results in an increase of cytoplasmic mRNA. The specific intron features responsible for efficient splicing and enhancement are not well characterized in plants. Internal deletions of up to 80% of two maize introns, Adh1 intron 1 and maize actin 3, indicate that large segments of these introns are dispensable for normal function. However, extensive deletion (> 75%) of Adh1 intron 1 diminishes both intron enhancement and splicing efficiency. This finding suggests that there are internal sequence motifs required for intron function, and that these motifs are redundant. We attempted to repair a deletion-impaired Adh1 intron 1 variant by adding back either oligomers of defined sequence content or fragments of maize internal intron sequence. The addition of AU-rich oligomers improved splicing efficiency and in one example, a U-rich oligomer activated a cryptic 3' splice acceptor. We also found that replacing the region proximal to the Adh1 intron 1 3' acceptor with U-rich sequence improved splicing. We found that adding G- and C-rich oligomers did not improve intron function, but a C-rich oligomer activated a cryptic 3' acceptor. The addition of internal intron sequence to an impaired intron improved splicing, and in one case, resulted in the activation of a cryptic 3' acceptor. We present evidence that U-rich sequence immediately upstream of the 3' splice junction increases splicing efficiency and contributes to, but does not uniquely specify, 3' acceptor AG choice.

Abstract

Transcription of the single-copy rice mitochondrial atp9 gene has been analyzed. We propose that there is a 0.65 kb primary transcript that is processed to an abundant 0.45 kb mRNA; a sequence motif at the 5' terminus of the 0.65 kb transcript shares 9 out of 11 nucleotides homology to the consensus promoter proposed for maize. There are several 3' termini based on RNase protection, and these termini map within or just distal to inverted repeats that could fold into a double stem-loop structure.

Abstract

The organization of the mitochondrial genome in the IR36 indica variety of rice was analyzed using constant-field and CHEF gel electrophoresis. The minimum complexity of the genome is estimated to be 300 kb. There was no evidence for a master circular molecule of this size. The genome exists in vivo in multiple subgenomic circular molecules or mitochondrial chromosomes. Using several rare-cutting restriction enzymes a restriction map was constructed for a 117-kb circular chromosome. Six genetic loci have been placed on this chromosome; cox1 and atp1 have been assigned precise positions while four additional genes (rrn26, cob2, atp6 and rrn18) were mapped to restriction fragments.

Abstract

Anthocyanin pathway-specific transcriptional activators R and C1 from the monocot maize were expressed in two dicots, Arabidopsis thaliana and Nicotiana tabacum. Expression of R caused augmented anthocyanin pigmentation in both plant species and augmented trichome (hair) production in Arabidopsis. Alone, C1 had no effect. Hybrid transgenic Arabidopsis expressing both C1 and R produced anthocyanins in root, petal, and stamen tissues that normally never express anthrocyanins. When R was expressed in the transparent testa glabrous (without anthocyanins and trichomes) mutant of Arabidopsis, the deficiency was complemented and both anthocyanins and trichomes were restored.

Abstract

Rice mitochondrial DNA contains an intact copy and a pseudogene copy of a apocytochrome b gene (cob-1 and cob-2, respectively). Using primer extension and capping analyses, the transcriptional start site has been mapped; an 11-base motif at the transcription start site closely matches the consensus promoter motifs proposed for maize, wheat and soybean mitochondrial genes. Although both copies are identical in the 5' upstream region and through most of the coding region, only cob-1-specific mRNA is detected on RNA gel-blots. Run-on transcription analysis indicates, however, that both cob-1 and cob-2 mRNAs are synthesized in vivo but less cob-2 is accumulated. At its mapped 3' terminus the cob-1 transcript possesses a sequence that could fold into a double stem-loop structure. The possible roles of a double stem-loop structure in mitochondrial gene expression are discussed.

Abstract

The 5'-untranslated leader of maize (Zea mays) heat-shock protein (hsp) 70 mRNA is required for translational competence during heat shock in protoplasts. When the beta-glucuronidase gene was used as a reporter mRNA, expression at elevated temperatures increased more than 10-fold when the hsp70 leader constituted the 5'-untranslated region. The hsp70 leader did not affect the physical half-life of the mRNA and, therefore, does not function at the level of transcript stability. The maize hsp70 leader was required to escape thermal repression in both maize and tobacco (Nicotiana tabacum) but was less effective in carrot. In addition, mRNAs containing the tobacco mosaic virus untranslated leader (omega) were also efficiently translated during heat shock, data suggesting that the presence of the omega sequence enables the transcript to escape the translational repression that occurs during thermal stress.

Abstract

Transposable element (TE) insertion into or near plant introns can cause intron skipping and alternative splicing events, resulting in reduced expression. To explore the impact of inserted sequences on splicing, we added non-intron sequence to two maize introns and tested these chimeric introns in a maize transient expression assay. Non-intron sequence inserted into Adh1-S intron 1 and actin intron 3 decreased expression from the luciferase reporter gene; the insertion sites tested were not in intron regions thought to be essential for splicing. Alternatively spliced mRNAs were not observed in transcripts derived from the insertion variants. In contrast, addition of an internal segment of an intron to Adh1-S intron 1 resulted in normal splice site selection and efficient processing. Because the normal intron sequence (including the conserved splice junctions) was retained in all constructs, we hypothesize that added non-intron sequence can interfere with intron recognition and/or splicing.

Abstract

The leader (called omega) of tobacco mosaic virus RNA enhances translation in both eukaryotes and prokaryotes. Although little secondary structure is predicted to exist within omega, the primary sequence of the 68 base leader is highly organized. Three copies of an eight base direct repeat and a (CAA)n region represent the two motifs found in the leaders of many TMV strains, and together these comprise 72% of omega. In previous deletion studies, no mutants exhibited loss-of-function, suggesting that functional redundancy exists within omega. We report here that a more comprehensive deletion analysis identified the motifs involved in translational enhancement. In a separate approach, oligonucleotides containing the sequence of each motif were used to construct leaders that varied in the number and configuration of the motifs. beta-Glucuronidase mRNA constructs containing these mutant leaders were synthesized in vitro and their translational efficiency measured in vivo following mRNA delivery to carrot protoplasts via electroporation. A combination of one copy of the 8 base direct repeat and a 25 base (CAA)n region was identified as the core regulatory element, although the (CAA)n motif is more critical. Two copies of the (CAA)n region are sufficient to confer a high level of enhancement and a leader composed of multiple copies of the direct repeat is moderately enhancing. Thus, these two motifs are functionally redundant.

Abstract

A large fraction of the transcripts of the Bronze-2 (Bz2) gene of maize (Zea mays L.) are unspliced in purple husk tissues. The accumulation of unspliced messages could have destructive potential if the intron-bearing mRNAs are translated into aberrant proteins. Our initial studies suggested that both genetic and physiological factors may influence the degree of splicing failure. Nuclear background rather than cis-sequence effects is shown to contribute to the genetic component. The accumulation of unspliced message does not appear to be directly influenced by diurnal effects on transcript abundance, by the expression level of the Bz2 gene, or by thermal stress. We also show that maize cell cultures (Black Mexican Sweet, BMS) can be used to examine the molecular details involved in splicing failure. Much like whole maize plants, the BMS cells excise the Bz2 intron with varying degrees of efficiency. In contrast with heterologous constructs containing plant introns, splicing of the native Bz2 intron can appproach 100% in BMS cells. Splicing of transcripts from a marked, introduced gene can be compared to the endogeneous Bz2 gene facilitating analysis of the impact of sequence changes.

Abstract

After epigenetic loss of Mutator activity, the family of Mu elements in Zea mays becomes immobile and highly methylated; in addition, Mu9, the presumptive autonomous regulatory element, is transcriptionally silent and its copy number decreases in successive crosses to non-Mutator lines. Spontaneous reactivation, scored as restoration of somatic instability of potentially mutable alleles of Bronze-2, of such cryptic Mutator lines is rare, occurring with a frequency of about 10(-4). Irradiation of pollen with 254 nm ultraviolet light increases reactivation rate in the progeny kernels by up to 40-fold. Accompanying reactivation, the copy number of Mu9 elements increased, two-fold in one line and 20 to 40-fold in a second line. Reactivation may involve direct DNA damage or immediate physiological stress in the treated pollen.

Abstract

ORF1 of the ubiquitous 2.3 kb linear plasmid of maize mitochondria encodes a 39 kDa protein detected with polyclonal antibodies raised to a beta-galactosidase:ORF1 fusion protein. Almost half of this protein is similar to a domain of the 130 kDa protein encoded by the S2 episome of mitochondria from cytoplasmic male-sterile lines. Antisera raised to the ORF1 2.3 kb plasmid product cross-reacts with the ORF1, 130 kDa protein from the S2 episome. Despite the shared domain, the proteins are differentially localized: the 130 kDa protein is membrane-associated while the ORF1 protein is found in the matrix. We discuss possible functions of the ORF1 protein.

Abstract

The putative maize transcription factor genes R and C1 are required for expression of reporter genes with promoters from the Bz1 and A1 genes, which encode enzymes required for anthocyanin biosynthesis in maize. Bz2 is another anthocyanin biosynthetic gene; we show that expression of a reporter gene from the Bz2 promoter also requires R and C1 when the fusion construct is introduced into maize kernels by particle gun bombardment. When electroporated into maize protoplasts from a suspension cell line not synthesizing anthocyanins, reporter genes with Bz2, Bz1, and A1 promoters are expressed only when both R and C1 expression plasmids are co-electroporated. Electroporation of R and C1 expression plasmids also induces the endogenous genes required for anthocyanin synthesis, resulting in pink protoplasts within 24 h. RNase protection analysis demonstrates that accumulation of mRNA from the endogenous Bz1 and Bz2 genes absolutely requires introduced R and C1. In time-course experiments there is a delay of 3-6 h before the Bz2 promoter is activated, supporting the proposed role for R- and C1-encoded proteins in transcriptional control. An excess of R relative to C1 suppresses expression of A1, Bz1, and Bz2 promoters, suggesting an interaction between the R and C1 proteins.

Abstract

Southern hybridization analysis using homologous maize probes indicated that orf25 and coxIII are closely linked in the mitochondrial genome of rice (Oryza sativa) cultivar IR36. The two coding regions were found on the same 5.1 kb BamHI fragment, and this fragment was cloned, mapped and partially sequenced. Using probes for each gene derived from the rice clone, a 2.4 kb dicistronic mRNA transcript was found containing both orf25 and coxIII coding regions. Multiple 5' ends were identified by primer extension analysis and a double stem/loop structure was mapped to the 3' end. The orf25 coding region shares greater than 85% identity with orf25 sequences from maize, tobacco and wheat, suggesting that orf25 may code for a conserved protein product.

Abstract

The structure of plant mitochondrial genomes has proven to be complex and difficult to study. Recombination across large and small repeated sequences can result in genome diversity within individual plants, as well as rapid evolutionary change in genome structure. The role of these repeats is becoming more obvious as mitochondrial genomes are examined in detail.

MUTATOR ACTIVITY IN MAIZE CORRELATES WITH THE PRESENCE AND EXPRESSION OF THE MU-TRANSPOSABLE ELEMENT MU9PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAHERSHBERGER, R. J., WARREN, C. A., Walbot, V.1991; 88 (22): 10198-10202

Abstract

Mutator is a powerful system for generating new mutants in maize. Mutator activity is attributable to a family of transposable, multicopy Mu elements, but none of the known elements is an autonomous (regulatory) element. This paper reports the discovery of Mu9, a 4942-base-pair Mu element that was cloned after it transposed into the Bronze-2 locus. Like other Mu elements, Mu9 has approximately 215-base-pair terminal inverted repeats and creates a 9-base-pair host sequence duplication upon insertion. A small gene family of elements that cross-hybridize to Mu9 has been found in all maize lines, and one of the other known Mu elements, Mu5, probably arose as a deletion of Mu9. Mu9 has several of the properties expected for the proposed regulator of Mutator activity. (i) The presence of Mu9 parallels the presence of Mutator activity in individuals from a line that genetically segregates for the Mu regulator. (ii) Lines that transmit Mutator to greater than 90% of their progeny have multiple copies of Mu9. (iii) Most maize lines that lack Mutator activity and that are not descended from Mutator lines lack the Mu9 element. (iv) Transcripts that hybridize to Mu9 are abundant in active Mutator lines, but they are absent from lines that have epigenetically lost Mutator activity. These correlations suggest that Mu9 is a candidate for the autonomous Mutator element.

Abstract

The 3'-untranslated region (UTR) of tobacco mosaic virus (TMV), which terminates in a tRNA-like structure, functionally substitutes for a poly(A) tail in both plant and animal cells. The addition of the TMV 3'-UTR to chimeric mRNA constructs increases their expression up to 100-fold, increasing both translational efficiency and mRNA stability. The domain largely responsible for the regulation maps to a 72 base region immediately upstream of the tRNA-like structure, however, the 3'-terminal, tRNA-like structure is required for full function. Its contribution is lost if separated from the upstream pseudoknot domain by as few as 5 bases or if 6 bases are removed from the 3'-terminus. Sequence addition to the 3'-terminus of the TMV 3'UTR or the upstream pseudoknot domain inhibits function in both tobacco and Chinese hamster ovary cells.

Abstract

Multiple genetic and epigenetic changes were detected within one plant generation at the bz2::mu1 mutable allele in a population of 118 plants. Loss of somatic instability in bz2::mu1 was usually correlated with methylation of the Mu1 transposable element; in 6 plants, somatic instability was lost as a result of mutations in bz2::mu1. This is a surprisingly high frequency of mutation per allele (2.5%) for the Mutator family, for which germinal revertants occur at a frequency of about 10(-4) per gamete. One germinal excision event was found that contained an 8 bp deletion, frameshift mutation in Bronze-2. The three other mutants described occurred as a result of abortive transposition, in which 75-77 bp deletions were generated at the junction between Bronze-2 and Mu1. We discuss the possible mechanisms, and the role of host factors in abortive transposition in maize.

Abstract

We have investigated whether reporter genes influence cytoplasmic regulation of gene expression in tobacco and Chinese hamster ovary (CHO) cells. Two genes, uidA encoding beta-glucuronidase (GUS) from Escherichia coli and Luc, encoding firefly luciferase (LUC), were used to analyze the ability of a cap, polyadenylated tail, and the 5'- and 3'-untranslated regions (UTR) from tobacco mosaic virus (TMV) to regulate expression. The regulation associated with the 5' cap structure and the TMV 5'-UTR, both of which enhance translational efficiency, was reporter gene-independent. The poly(A) tail and the TMV 3'-UTR, which is functionally equivalent to a poly(A) tail, increase translational efficiency as well as mRNA stability. The regulation associated with these 3' ends was highly reporter gene-dependent; their effect on GUS expression was almost an order of magnitude greater than that on LUC expression. In tobacco, the tenfold reporter gene effect on poly(A) tail or TMV 3'-UTR function could not be explained by a differential impact on mRNA stability; GUS and LUC mRNA half-life increased only twofold when either the poly(A) tail or TMV 3'-UTR was present. In CHO cells, however, GUS mRNA was stabilized to a greater extent by a poly(A) tail or the TMV 3'-UTR than was LUC mRNA.

Abstract

The organization of the mitochondrial genome in plants is not well understood. In maize mitochondrial DNA (mtDNA) several subgenomic circular molecules as well as an abundant fraction of linear molecules have been seen by electron microscopy. It has been hypothesized that the circular molecules are the genetic entities of the mitochondrial genome while the linear molecules correspond to randomly sheared mtDNA. A model has been proposed that explains the mechanism of generation of subgenomic circles (of a predictable size) by homologous recombination between pairs of large direct repeats found on a large (approximately 570 kb for the fertile (N) cytoplasm) master circle. So far the physical entities of the mitochondrial genome, as they exist in vivo, and the genes they carry, have not been identified. For this purpose, we used two gel systems (pulsed field gel electrophoresis and Eckhardt gels) designed to resolve large DNA. Large DNA was prepared from the Black Mexican Sweet (BMS) cultivar. We resolved several size classes of mtDNA circles and designate these as chromosomes. A 120 kb chromosome was mapped in detail. It is shown to contain the three ribosomal genes (rrn26, rrn18 and rrn5) plus two genes encoding subunits of cytochrome oxidase (Cox1 and Cox3); it appears to be colinear with the 570-kb master circle map of another fertile cytoplasm (B37N) except at the "breakpoints" required to form the 120-kb circle. The presence of the 120-kb chromosome could not have been predicted by homologous recombination through any of the known repetitive sequences nor is it a universal feature of normal maize mitochondria. It is present in mitochondria of BMS suspension cultures and seedlings, but is not detectable in seedlings of B37N. No master genome was detected in BMS.

Abstract

Germinal and somatic excision products of Mu1 from the insertion allele bz::mu1 were selectively amplified from maize cob tissue. The sequence of these "footprints" often included deletions at the target site, suggesting that substantial exonucleolytic degradation occurs upon excision of the element. In addition to deletions of target site sequences, single base insertions were also found. The isolation of an excision product including a 4 bp inverted duplication of the target site provides evidence that the double-stranded chromosomal break generated by Mu excision may be terminated by a covalently closed hairpin structure. The majority of excision products, however, do not include inverted duplications of target site sequences, suggesting that such structures are the result of occasional repair activities, rather than an essential step in the mechanism of Mu excision. The sequence of the Mu insertion sites of the bz::mu1 and bz::mu2 alleles is also presented.

Abstract

The B locus in maize is required for the accumulation of anthocyanin pigments. Numerous B alleles have been described: each determines a particular pattern of pigment synthesis with respect to the tissues that are pigmented and the time during development that pigment synthesis begins. We report here a genetic analysis of one B allele, B-Peru, which regulates synthesis of pigments in both kernel and plant tissues. We used stocks with active Mutator transposable elements to produce eight mutations in B-Peru. All eight alter pigment synthesis in all the kernel and plant tissues pigmented by B-Peru, suggesting that each mutation has disrupted a region of the gene required for expression in all tissues. Six of the mutations cause a colorless phenotype, while two cause a reduction in pigment in both kernel and plant tissues. Four of the mutations are unstable, and four are stable upon self-pollination. Multiple independent revertants were isolated from each unstable allele. DNA blot analysis demonstrated that all eight mutants are the result of insertions within an approximately 5-kb region that encodes the B-Peru transcript. One of the four unstable alleles contains a Mu element, Mu1.7. Two others contain insertions related to the Spm family of transposable elements. Thus, our Mutator stocks also contained active transposable elements from the unrelated Spm family. Our experiments suggest that the B-Peru allele is not complex, but contains a single coding region that regulates pigment synthesis in multiple tissues.

INTRON ENHANCEMENT OF GENE-EXPRESSION AND THE SPLICING EFFICIENCY OF INTRONS IN MAIZE CELLSMOLECULAR AND GENERAL GENETICSLuehrsen, K. R., Walbot, V.1991; 225 (1): 81-93

Abstract

The inclusion of the alcohol dehydrogenase 1-S(Adh 1-S) intron 1 in the transcription unit of maize gene constructs has been shown to increase gene expression in cultured maize cells. We have extended these studies with Adh1-S intron 1 using the firefly luciferase, Escherichia coli beta-glucuronidase and chloramphenicol acetyltransferase reporter genes adjoined to different plant promoters and find enhancement of transient gene expression in all cases but one. We also show that the enhancement phenomenon can be mediated by the third intron of the maize actin gene. In all cases tested, the inclusion of an intron results in increased levels of steady-state RNA. The degree of enhancement depends on the exon sequences flanking the intron; flanking exons also influence the efficiency of intron splicing. Unexpectedly, unspliced RNAs accumulate during the transient assay.

Abstract

Lysed maize mitochondria synthesize RNA in the presence of radioactive nucleoside triphosphates, and this assay was utilized to compare the rates of transcription of seven genes. The rates of incorporation varied over a 14-fold range, with the following rank order: 18S rRNA greater than 26S rRNA greater than atp1 greater than atp6 greater than atp9 greater than cob greater than cox3. The products of run-on transcription hybridized specifically to known transcribed regions and selectively to the antisense DNA strand; thus, the isolated run-on transcription system appears to be an accurate representation of endogenous transcription. Although there were small differences in gene copy abundance, these differences cannot account for the differences in apparent transcription rates; we conclude that promoter strength is the main determinant. Among the protein coding genes, incorporation was greatest for atp1. The most active transcription initiation site of this gene was characterized by hybridization with in vitro-capped RNA and by primer extension analyses. The DNA sequences at this and other transcription initiation sites that we have previously mapped were analyzed with respect to the apparent promoter strengths. We propose that two short sequence elements just upstream of initiation sites form at least a portion of the sequence requirements for a maize mitochondrial promoter. In addition to modulation at the level of transcription, steady-state abundance of protein-coding mRNAs varied over a 20-fold range and did not correlate with transcriptional activity. These observations suggest that posttranscriptional processes are important in the modulation of mRNA abundance.

Abstract

Maize transposable elements, when inserted in or near genes, alter expression by several transcriptional and post-transcriptional mechanisms. Three independent, unstable insertions of the transposable element Mutator (Mu) into the first intron of the Alcohol dehydrogenase-1 (Adh1) gene have been shown to decrease expression [Strommer et al. (1982). Nature 300, 542-544]. We have developed an approach to elucidate the underlying molecular mechanisms responsible for the mutant phenotypes. Mu1 elements were inserted into Adh1-S intron 1 in vitro to create plasmid facsimiles of the mutant alleles. The Mu1 element was also inserted at novel positions within intron 1 to create new mutations. The Mu1/intron constructions were placed between the Adh1-S promoter/exon 1 segment and a reporter gene (firefly luciferase or beta-glucuronidase), and these chimeric gene constructs were tested in transient assays in maize protoplasts. When compared with the appropriate control, the Mu1 insertions decreased reporter gene expression to levels approximating the alcohol dehydrogenase enzyme activities observed for the Adh1-S mutants in vivo. The Mu1 insertions also showed a polarity effect with luciferase expression increasing as the insertions were placed nearer the 3' splice junction. In addition, Mu1 insertions within a different intron, actin intron 3, also significantly reduced luciferase expression, indicating that Mu1 insertions within introns are likely to diminish expression in many genes. The presence of the Mu1 sequences was correlated with decreased levels of steady-state luciferase transcript. Deletion analysis of the Mu1 element and RNase mapping indicate that the transposable element contains RNA processing signals in its central region that are largely responsible for the decrease in expression.

Abstract

The maize Bronze-2 (Bz2) gene, whose product acts late in the anthocyanin biosynthetic pathway, has been cloned and its transcript has been mapped. We have developed a general procedure for reconstructing wild-type alleles from transposable element-induced mutants. An existing transposon-containing clone, bz2::mu1 [McLaughlin, M., and Walbot, V. (1987). Genetics 117, 771-776], was modified by replacing the region of bz2::mu1 containing the transposon with the corresponding polymerase chain reaction-amplified sequence from the progenitor allele that has no Mu insertion. Particle gun delivery of the reconstructed Bz2 gene to embryonic scutellar tissue lacking a functional Bz2 gene complemented the bz2 mutant phenotype, as demonstrated by the production of purple spots. Having cloned the wild-type allele, we then analyzed the Bz2 transcript, whose features include an 82-nucleotide 5'-untranslated leader, one small intron (78 base pairs) within the coding region, and multiple polyadenylation sites. Four Mutator transposon insertions that eliminate gene function were mapped within the 850-nucleotide transcription unit. We found that variable levels of unspliced Bz2 RNA are present in purple husk tissue; this finding may indicate that the expression of Bz2 is regulated in part at the level of transcript processing.

Abstract

The genomes of many RNA viruses terminate in a tertiary structure similar to the L-conformation of tRNAs and this structure is recognized by many tRNA-specific enzymes such as aminoacyl-tRNA synthetase. Virtually the entire 3'-untranslated region (UTR) of tobacco mosaic virus (TMV) RNA is involved in an extended tertiary structure containing, in addition to a tRNA-like structure, a pseudoknot domain that lies immediately upstream. Although the functions of these structures are not well understood, they are essential to the virus. We demonstrate that the addition of the 204-base TMV 3'-untranslated region to foreign mRNA constructs can increase gene expression up to 100-fold compared to nonadenylated mRNA. The 3'-UTR of TMV was equal to or greater than a polyadenylated tail in enhancing gene expression in electroporated dicot and monocot protoplasts. The TMV 3'-UTR is functionally similar to a polyadenylated tail in that it increases mRNA stability and translation and must be positioned at the 3' terminus to function efficiently. Similar effects on expression were observed in Chinese hamster ovary cells, demonstrating that the sequence functions in a wide range of eukaryotes. When the extended tertiary structure was dissected, the upstream pseudoknot domain was found to be largely responsible for increasing expression. The inclusion of the tRNA-like structure, however, was important for full regulation.

Abstract

Using a battery of methylation-sensitive restriction enzymes, cytosine methylation at 23 sites in a 7.6 kb region surrounding the Alcohol dehydrogenase-1 (Adh1) gene was measured in DNA prepared from immature maize cobs. Both the 5' upstream region and the entire coding region were hypomethylated in the two alleles examined. Methylation in Adh1 is independent of changes in Mutator transposable element methylation. The role of DNA methylation in Adh1 gene regulation is discussed.

Abstract

The ability of transposable elements (TEs) to insert into or excise out of a genetic locus can be regulated by genetic, environmental, and developmental factors. Tissue- or organ-specific activity of TEs is a frequent and well-characterized example of spatial, developmental regulation. Regulation of the timing of TE activity during ontogeny is less well understood. To analyze timing, TE-induced variegation was quantified in the aleurone of maize kernels, a tissue composed of only a single layer of cells, and sector sizes were assigned to specific cell divisions in aleurone development. Three TE families, Mu, Spm, and Ac/Ds, were studied at two genetic loci. It was found that the frequency of transposon excision changes drastically (up to 30-fold increase or equivalent decrease) during the proliferation of the aleurone. Moreover, these changes occur at the same cell divisions in all three TE families. These results suggest that the timing of TE excision during maize development can be controlled by the host.

Abstract

Analysis of the 18 S and 5 S ribosomal RNA transcripts of maize mitochondria is described. The 5' and 3' ends of both mature rRNAs were defined by S1 nuclease protection analysis, which also showed that a small fraction of the total 18 S and 5 S rRNA population resides on common transcripts. The 5' termini of many of these RNAs are upstream from the mature 18 S 5' endpoint. Northern hybridization detected several high molecular weight RNAs that were homologous to the 5 S and 18 S genes and their flanking sequences. Because the two rRNAs share a number of transcripts, we propose that these genes are transcribed as one or more large dicistronic RNAs that are subsequently processed to mature ribosomal RNA molecules. A model for the processing of the large putative precursors is presented.

Abstract

We present a model for the kinetics of methylation and demethylation of eukaryotic DNA; the model incorporates values for de novo methylation and the error rate of maintenance methylation. From the equations, an equilibrium is reached such that the proportion of sites which are newly methylated equals the proportion of sites which become demethylated in a cell generation. This equilibrium is empirically determined as the level of maintenance methylation. We then chose reasonable values for the parameters using maize and mice as model species. In general, if the genome is either hypermethylated or hypomethylated it will approach the equilibrium level of maintenance methylation asymptotically over time; events occurring just once per life cycle to suppress methylation can maintain a relatively hypomethylated state. Although the equations developed are used here as framework for evaluating events in the whole genome, they can also be used to evaluate the rates of methylation and demethylation in specific sites over time.

Abstract

Active Mutator lines of maize (Zea mays L.) have a high mutation rate and contain multiple hypomethylated 1.4-kb and 1.7-kb Mu transposable elements. Correlated with the inactivation of the Mutator system, these Mu elements cease to transpose and become more methylated. To determine whether the shock of tissue culture can affect Mutator activities, F1 progenies of outcrosses between active or inactive Mutator stocks and inbred line A188 were used to initiate embryogenic callus cultures. HinfI restriction digestion of genomic DNA isolated from 3-5-month-old cultures demonstrated that there is a very good correlation between the modification state of Mu elements in the cultures and the Mutator parent. Despite the dedifferentiation and rapid proliferation characteristic of tissue culture, the Mutator activity state is relatively stable during an extended tissue culture period. Cultures established from inactive Mutator lines were not reactivated; cultures established from active lines maintained a high Mu copy number, and most Mu elements remained unmodified. In contrast, weakly active Mutator parents gave rise to cultures in which Mu element modification could switch between low and high methylation during the culture period. Evidence for transposition was investigated with EcoRI digestion of genomic DNA isolated at different times during culture. The appearance of novel Mu-hybridizing fragments and a strong background hybridization are interpreted as evidence that transposition events occur during culture. Plants regenerated from such active cultures transmitted Mutator activity to their progeny.

Abstract

The effects of cold on protein and RNA metabolism in leaves of rice (Oryza sativa L.) seedlings were investigated. Treatment with a diurnal cycle of 15/10 degrees C or 11/6 degrees C for up to 1 week resulted in progressive changes in the protein synthesis pattern after in vivo labeling of intact rice leaves with [(35)S]methionine. These changes were reversed when the seedlings were returned to normal growth temperatures. While de novo accumulation of several abundant proteins was suppressed, some polypeptides were consistently found to be cold-induced. Synthesis of ribulose 1,5-bisphosphate carboxylase (Rubisco) was drastically reduced after 7 days of cold. Using immunoprecipitation of Rubisco, evidence was obtained that the suppression was greater for the small subunit (over 90%) than for the large subunit (80%), indicating a partial loss of coordination in their synthesis. Preformed Rubisco as well as other cold-suppressed proteins were stable for up to 7 days at 11/6 degrees C. Cold-sensitive rice cultivars responded with similar but more drastic changes in the protein synthesis pattern when compared to cold-tolerant varieties. The suppression of Rubisco synthesis by cold was shown to result from reduced levels of the mRNAs encoding both subunits; their decrease paralleled the lower protein synthesis of each. The levels of other chloroplast-encoded mRNAs, especially psaB, and of the nuclear encoded chlorophyll a/b binding protein, also strongly decreased in the cold, whereas the transcripts of the mitochondrial genes apt9, coxIII, and most nuclear genes analyzed were unaffected or only slightly reduced. These data indicate that some chloroplast functions are disturbed during cold stress. One nuclear gene known to be induced by water stress and ABA (Rab21) was also found to be induced by cold treatment.

Abstract

The nucleotide sequence and transcription pattern of the linear 2.3 kb plasmid of maize mitochondria was analyzed in order to elucidate its possible function in the organelle. The plasmid has 170 bp inverted repeats at its termini composed, in turn, of shorter repetitive sequences. An open reading frame within the plasmid is transcribed and can potentially specify a 33 kD product. In addition the plasmid contains two tRNA genes homologous to chloroplast sequences; the tRNApro(CAA) and the tRNAtrp(UGG). Both of the tRNA genes of the plasmid are transcribed, but apparently only the tRNAtrp is processed to the correct size. These tRNA sequences are found in the main mitochondrial genome of all higher plants tested, and in most maize relatives. An exception is the close maize relative Northern teosinte in which the tRNAtrp gene is also carried on a plasmid. These results suggest that the 2.3 kb plasmid has acquired the tRNA sequences from the main mitochondrial DNA. It is possible that the plasmid-encoded tRNAtrp gene is essential for organelle function thereby ensuring the maintenance of the plasmid in the mitochondrion.

Abstract

We examined the role of alcohol dehydrogenase (ADH) in the metabolism and survival of hypoxic maize (Zea mays L.) root tips. The dependence of the rate of ethanolic fermentation, cytoplasmic pH, and viability on the activity of ADH in maize root tips during extreme hypoxia was determined. Maize lines with ADH activities differing over about a 200-fold range were studied. Effects of genetic background were controlled by comparing pairs of F4 progeny of crosses between mutant (low ADH activity) and reference inbred lines. The capacity of hypoxic root tips to perform ethanolic fermentation exhibited a dependence on ADH activity only at activities found in Adh 1 nulls. The ability of maize root tips to withstand prolonged and extreme hypoxia was like-wise independent of ADH activity, except at the lowest activities. Root tips that exhibited lower tolerance of hypoxia had more acidic cytoplasm during extreme hypoxia. We conclude that the activity of ADH in normal maize root tips does not limit the capacity for energy production via fermentation, and does not determine viability under extreme hypoxia. The significance of the induction of ADH activity in plants by hypoxia is discussed.

Abstract

By direct RNA mapping, we have identified the precise 5' end of the maize mitochondrial 26S rRNA. The 5' termini of the 26S rRNA are 17 and 18 nt downstream from the 5' end predicted by Dale et al. (1984) from homology between the maize rRNA and E. coli large subunit ribosomal RNA. In addition, we have discovered a larger 26S species, presumably a 26S precursor, and precisely mapped its 5' end. The maize mitochondrial rRNA genes are probably regulated by control regions that differ from those already characterized in other genomes: the maize mitochondrial 26S, 18S and 5S rRNAs lack sequences that resemble the promoter regions of genes from other mitochondria and bacteria. However, the mature and precursor rRNAs all contain a tetranucleotide, AAUC, at their 5' ends (AAAC in the 5S rRNA). The sequence is also seen in the same position in several other plant mitochondrial mRNAs. We propose therefore that AAUC is a transcription or processing signal which is possibly unique to plant mitochondria.

Abstract

In this paper we demonstrate that RNA sequences present upstream and downstream of a reporter gene coding region play an important role in determining the amount of protein produced from an mRNA. A translational enhancer, omega, derived from tobacco mosaic virus, when present at the 5'-end of beta-glucuronidase mRNA increased the efficiency of translation 16-fold to 18-fold in electroporated tobacco or carrot protoplasts, and threefold to 11-fold in maize or rice protoplasts. The presence of omega did not alter the half-life of the mRNA in vivo. We also demonstrate for the first time that a minimum polyadenylated tail length of 25 adenylate residues is sufficient to substantially increase the expression and half-life of the reporter mRNA in plants. When in vitro-produced mRNAs were synthesized such that extra sequence was added to the 3'-end of the poly(A) tail, however, the final level of expression was decreased up to 80%. Omega, the translational enhancer, and a poly(A) tail function independently of each other; their combined effect on translation, when both are present in an mRNA, is the multiplication of their individual effects. Histochemical analysis for the presence of beta-glucuronidase in tobacco established that virtually all viable cells receive mRNA during electroporation. Video image analysis of tobacco protoplasts electroporated with luciferase mRNA demonstrated that there is a wide range in the level of expression of this marker. Carrier RNA, when present during electroporation, had only a modest effect on increasing mRNA uptake. Reporter mRNA expression in electroporated protoplasts was directly proportional to the input mRNA up to at least 30 micrograms/ml.

Abstract

Expression of a 130 kDa protein from open reading frame 1 of the integrated form of the R2 mitochondrial plasmid in normal mitochondria of B37 and other inbred lines is described. The protein appears identical to that synthesized by the closely related S2 episome found in cytoplasmic male sterile maize of the S type. Protein was detected using antisera raised against a beta-galactosidase:ORF1 fusion product containing the most antigenic region of the ORF1 product. Detection of this protein is in contrast to previous reports that mitochondria of normal, male-fertile lines either do not contain this protein, or that there are 11 in-frame stop codons in the reading frame. The integrated R2 of B37N was cloned and this region sequenced, confirming that a continuous open reading frame existed. These results are discussed in relation to the possible role of the S-type episomes in causing cytoplasmic male sterility.

Abstract

The regulation of excision of Mu elements of the Mutator transposable element family of maize is not well understood. We have used somatic instability of Mu receptor elements from the Bronze 1 and Bronze 2 loci to monitor the frequency and the timing of excision of Mu elements in several tissues. We show that spot size in the aleurone of a bz2::mu1 stock varies between one to approximately 256 cells. This indicates that excision events begin eight divisions prior to full aleurone differentiation and end after the last division of the aleurone. We show that excision is equally biased for late events in all other tissues studied. A locus on chromosome 5 has been identified that affects spot size, possibly by altering the timing of Mu excision. Using somatic excision as an assay of Mutator activity, we found that activity can change in small sectors of the tassel; however, there are no overall activity changes in the tassel during the period of pollen shedding. We also report the recovery of germinal revertants for the bz1::mu1 and bz2::mu1 alleles. One of these revertant alleles was characterized by Southern blot analysis and found to be similar to the progenitor of the mutable allele.

Abstract

The response of maize (Zea mays L.) protoplasts to high temperature stress was investigated. After isolation and electroporation, protoplasts were preincubated for 12 hours at 26 degrees C then incubated for 6 hours at elevated temperatures. The pattern of polypeptides synthesized by these protoplasts during the last hour was monitored by in vivo labeling with (35)S-methionine. Incubation at 40 degrees and 42 degrees C resulted in the synthesis of polypeptides not detectable at 26 degrees C. Introduction of a chimeric maize heat shock protein 70 promoter-chloramphenicol acetyltransferase coding region gene into protoplasts via electroporation resulted in the temperature-dependent induction of chloramphenicol acetyltransferase activity with maximal activity at 40 degrees C. In the same protoplasts, a second chimeric gene, in which the firefly luciferase coding region was under the control of the 35S promoter from cauliflower mosaic virus, did not show an increase in expression after incubation at higher temperatures. Maize protoplasts provide a system to study molecular responses to high temperature stress.

NUMEROUS TRANSCRIPTION INITIATION SITES EXIST FOR THE MAIZE MITOCHONDRIAL GENES FOR SUBUNIT-9 OF THE ATP SYNTHASE AND SUBUNIT-3 OF CYTOCHROME-OXIDASEPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAMulligan, R. M., Lau, G. T., Walbot, V.1988; 85 (21): 7998-8002

Abstract

Transcripts for plant mitochondrial genes are frequently present as multiple size classes. In maize, these differences often result from variation in the 5' noncoding region. To determine where transcription initiates, primary (unprocessed) transcripts were specifically labeled in vitro by the capping reaction catalyzed by guanylyltransferase. Direct mapping of transcription initiation sites was accomplished by hybridization of in vitro-capped RNA with the 5' flanking sequences of mitochondrial genes and subsequent digestion with single-strand-specific RNases. The RNase protection experiments identified three transcription initiation sites for subunit 3 of cytochrome oxidase and at least six transcription initiation sites for subunit 9 of ATP synthase. Thus, transcript size heterogeneity is primarily the result of multiple transcription initiation sites for these genes rather than RNA processing. Primer extension analyses of maize mitochondrial RNA were used to precisely establish the sequences at the initiation sites. Comparison of sequences at transcription initiation sites suggests that some homology exists at these sites, although no highly conserved consensus sequence is obvious.

Abstract

The omega sequence at the 5'-terminus of tobacco mosaic virus (TMV) RNA acts as a translational enhancer. The differential in omega-associated translational enhancement between the in vitro translation system derived from wheat germ (WG) and that from rabbit reticulocytes (MDL) was exploited to identify that lysate component which was responsible for a lysate's characteristic response to omega. Using fractionated MDL and WG lysates, which were reconstituted in various combinations, the high salt-washed ribosomal fraction was determined to be the responsive element in a lysate. Analysis of omega's ability to enhance translation was greatest at low mRNA and high ribosomal concentrations and to occur in the early phase of an in vitro translation assay. Translation of omega-containing CAT mRNA was more sensitive to the presence of micrococcal nuclease than CAT mRNA without an omega. In substitution experiments, WG ribosomes functioned at much reduced efficiency in MDL as did MDL ribosomes in WG lysate. The initiation factor-containing fraction of one system could not, as a whole, functionally replace that of the other and actually acted to inhibit translation in the heterologous system.

Abstract

Variation in the length of the 5' non-coding region of mitochondrial gene transcripts could result from multiple transcription initiation sites or post-transcriptional processing events. To distinguish between these possibilities, we have utilized the in vitro capping reaction catalyzed by guanylyl transferase to specifically label the 5' end of primary, unprocessed transcripts. Hybridization of in vitro capped mtRNA to immobilized DNA from the 5' flanking regions of 26 S, 18 S and 5 S rRNA genes and two protein-coding genes, ATP synthase subunit 9 (atp9) and apocytochrome b (cob), identified regions where transcription initiates. Single-strand specific RNase treatment of in vitro capped RNA hybridized to immobilized DNA containing the 5' flanking sequences from cob and atp9 suggests that these genes have multiple transcription initiation sites. Direct mapping of transcription initiation sites for the rRNA genes indicated that single major transcription initiation sites exist at approximately 180 and 230 nucleotides upstream from the mature 26 S and 18 + 5 S rRNA genes, respectively. Labeling of processed transcripts bearing a 5' hydroxyl moiety with T4 polynucleotide kinase and subsequent hybridization to the rRNA genes indicated that the mature forms of the rRNA are processed.

Abstract

We discuss the properties of the Mutator (Mu) transposable element family of maize. We report the cloning of bz2-mu1, a mutable allele containing a 1.4-kb Mu element, using a combination of transposon tagging and tests for differential hybridization to northern and Southern blots. We report the sequence of this allele and the Mu element insertion, and propose a model for the structure of the Bz2 locus. We discuss the relationship between increased DNA modification of Mu elements and loss of somatic instability at bz2-mu1. To further explore this aspect of regulation of Mutator, we have used gene-specific probes to determine the level of modification at this locus in active and inactive Mutator lines. We have also utilized CsCl density gradients to estimate the overall level of DNA modification in active and inactive lines; we find that Mu elements in active lines are hypomethylated relative to other maize nuclear DNAs examined, and that in inactive lines the level of modification in Mu elements is similar to the genome as a whole. Utilizing gamma-irradiation, we have demonstrated that inactive lines can be reactivated; this reactivation is first noted as restitution of the spotted kernel phenotype characteristic of bz2-mu1 in active Mutator lines. Hybridization analysis of DNA from reactivated plants demonstrates that the Mu elements in general, and specifically the Mu element at bz2-mu1, have the lower level of DNA modification characteristic of active lines. These results are discussed in terms of the role and timing of DNA modification in regulating Mutator activities.

Abstract

Maize nuclear DNA sequences capable of promoting the autonomous replication of plasmids in yeast were isolated by ligating Eco RI-digested fragments into yeast vectors unable to replicate autonomously. Three such autonomously replicating sequences (ARS), representing two families of highly repeated sequences within the maize genome, were isolated and characterized. Each repetitive family shows hybridization patterns on a Southern blot characteristic of a dispersed sequence. Unlike most repetitive sequences in maize, both ARS families have a constant copy number and characteristic genomic hybridization pattern in the inbred lines examined. Larger genome clones with sequence homology to the ARS-containing elements were selected from a lambda library of maize genomic DNA. There was typically only one copy of an ARS-homologous sequence on each 12-15 kb genomic fragment.

Abstract

There maize nuclear DNA fragments were isolated on the basis of their ability to confer replication on chimeric plasmids in yeast. These Eco RI fragments of 2.5, 2.8 and 5.5 kb are repeated elements within the maize genome. The 2.5 and 2.8 kb fragments represent a family of elements repeated 11 000 times in the maize haploid genome, while the 5.5 kb fragment is part of another family of 28 000 elements. These fragments were subcloned to further define the unique region of ARS activity. The sequence of each 550-650 bp ARS subclone is reported here, and compared to the flanking regions which do not show ARS activity. The ARS elements are 65-70% A+T as compared to 50-55% for the maize genome as a whole. There is approximately 15% sequence divergence, as well as variation of ARS efficiency, among family members. ARS subclones contain the proposed yeast consensus sequence.

Abstract

In the progeny of an active Mutator plant, the number of Mu elements increases on self-pollination and maintains the average parental Mu content on outcrossing to a non-Mutator line; both patterns of transmission require an increase in the absolute number of Mu elements from one generation to the next. The same average copy number of Mu elements is transmitted through the male and female, but there is wide variation in the absolute copy number among the progeny. In inactive Mutator plants-defined both by the loss of somatic instability at a reporter gene (bronze2-mu1) and by modification of the HinfI sites in the terminal inverted repeat sequences of Mu elements - the absolute copy number of Mu elements is fixed in the parent. Thus, in outcrosses Mu element number is halved, and on self-pollination Mu copy number is constant. Reactivation of somatic mutability at cryptic bz2-mu1 alleles in inactive individuals by crossing to an active line seems not to involve an increase in Mu element copy number transmitted by the inactive individual. These and other results suggest that increases in Mu copy number occur late in plant development or in the gametophyte rather than after fertilization.

Abstract

Sequences of Bronze2 (Bz2), a maize gene which is required for the synthesis of the purple pigment anthocyanin, have been cloned by combining the techniques of transposon tagging and differential hybridization. First, a mutable bz2 allele (bz2-mu1) was recovered from a Mutator line. The mutation was assumed to result from an insertion of the transposable element Mutator (Mu), which is found in 10-60 copies in Mutator lines. A library was prepared using DNA isolated from bz2-mul, and a small bank of Mu-specific candidate clones was selected. Because much is known about the genetic requirements for the synthesis of anthocyanin in different tissues, we were able to identify the bz2 clone based on its hybridization to RNA isolated from different bz2 mutants. Only one clone hybridized to RNA isolated from the husks of purple (Bz2, B, Pl) plants but not to RNA isolated from the husks of green (an-bz2-6923, b, pl) plants. Further confirmation was provided by the hybridization pattern of the clone on blots containing DNA from other bz2 mutants. We present a restriction map of the clone, indicating the location and orientation of the 1.4-kbp Mu insert. We identify the transcribed region, the direction of transcription and the location of the 1.4-kbp Mu insert in an independently isolated mutant, bz2-mu2.

Abstract

Using electroporation-mediated gene transfer, the gene encoding the Slow (S) migrating polypeptide of the maize (Zea mays L.) alcohol dehydrogenase-1 (Adh1) enzyme has been introduced stably and transiently into maize cells containing an endogenous Fast (F) ADH1 electromorph. In stable transformants an 11.5-kb fragment was sufficient to program normal S expression relative to the endogenous F allele. In transient assays, Adh1-S gene constructs lacking the 9 Adh1-S intervening sequences (introns) were expressed at levels 50- to 100-fold less than the intact gene; the presence of intron 1 alone restored levels of gene expression to those found with the intact gene. The last two introns also stimulate Adh1-S expression, but the level is threefold below that of the intact gene. The expression of a chimeric chloramphenicol acetyltransferase (CAT) gene utilizing the 5' promoter and 3' polyadenylation regions of the Adh1 gene was increased 100-fold by the addition of sequences containing the Adh1 intron 1. The Adh1 intron 1 sequences did not stimulate CAT expression when located outside the transcribed region. When located within the transcribed region, the Adh1 intron 1 region efficiently stimulated CAT expression only when located between the promoter and the CAT coding region. A construct containing the Adh1 intron 1 fragment produced 40-fold more mRNA than a construct containing an equivalent cDNA fragment. Both the Adh1 intron 1 and the intron from a second maize gene, Bronze1, stimulated expression from other promoters (cauliflower mosaic virus 35S and nopaline synthase) and of other coding regions (luciferase and neomycin phosphotransferase II) as well. These results indicated that introns increase both Adh1 and chimeric gene expression in maize and the optimal location for such an intron is near the 5' end of the mRNA.

S2 EPISOME OF MAIZE MITOCHONDRIA ENCODES A 130-KILODALTON PROTEIN FOUND IN MALE STERILE AND FERTILE PLANTSPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAZabala, G., OBRIENVEDDER, C., Walbot, V.1987; 84 (22): 7861-7865

Abstract

The mitochondrial genome of the S-type male-sterile cytoplasm of maize contains two linear episomes, S1 (6397 base pairs) and S2 (5453 base pairs). The S2 episome contains two large unidentified open reading frames, URF1 (3512 base pairs) and URF2 (1017 base pairs). We have demonstrated that a polypeptide with an apparent molecular mass of 130 kDa is the gene product of URF1. This polypeptide was first detected in Coomassie blue-stained protein gels of cms-S (where cms = cytoplasmic male sterile) but not in those of cms-T, cms-C, or normal mitochondrial proteins. The protein product of a translational fusion containing the 5' end of Escherichia coli lacZ and an internal segment from URF1 of S2 was recognized by antisera raised against the 130-kDa variant polypeptide. The mitochondria of fertile F(1) hybrids of cms-S x Ky21 (the male parent carrying nuclear fertility restoration genes) contain as much of the 130-kDa protein as is found in cms-S mitochondria of sterile plants. Spontaneous fertile cytoplasmic revertants from cms-S in a WF9 nuclear background also synthesized the 130-kDa polypeptide. Therefore, the mere presence or absence of the URF1 gene product of S2 does not determine the fertility status of maize plants, because male sterile and male fertile (nuclear restored and revertant) plants can contain equivalent amounts of the 130-kDa polypeptide.

ISOLATION AND CHARACTERIZATION OF A 1.7-KB TRANSPOSABLE ELEMENT FROM A MUTATOR LINE OF MAIZEGENETICSTaylor, L. P., Walbot, V.1987; 117 (2): 297-307

Abstract

We have cloned and sequenced a 1.7-kb Mu element from a Mutator line of maize and compared its structure to Mu1, a 1.4-kb element. With the exception of a 385-bp block of DNA present in the 1.7-kb element, these transposable elements are structurally similar, sharing terminally inverted and internal direct repeated sequences. Derivation of 1.4-kb elements from the 1.7-kb class via deletion of internal sequence is suggested by the finding that a portion of the extra DNA in Mu1.7 is part of a truncated direct repeat sequence in the 1.4-kb element. An abundant poly(A)+ RNA homologous to a portion of this extra DNA is present in several tissues of both Mutator and non-Mutator lines. Analysis of transcripts from an unstable mutant bronze 1 (bz) allele containing a Mu1.7 element inserted in an exon of the gene detects three species of poly(A)+ RNA that hybridize to a Bz1 (Bronze) gene probe: the largest contains the entire Mu1.7 element in the Bz1 gene transcript; another appears to be a spliced, chimeric transcript; the smallest is normal size Bz1 mRNA. The latter is most likely encoded by the normal-size alleles detected by Southern analysis of tissue expressing purple pigment, suggesting that normal gene function is restored by excision of the Mu1.7 element.

Abstract

A general method to introduce RNA molecules into plant protoplasts and animal cells is described. This technique utilizes the ability of electric pulses of high field strength to form pores in biomembranes. RNA molecules containing the coding region for the bacterial enzyme chloramphenicol acetyltransferase (CAT) were used as a model system. The presence of CAT activity as a result of the in vivo translation of the introduced RNA is entirely dependent on the presence of a 5' cap and greatly increased by the presence of a poly A tail at the 3' end. The introduction of RNA into eukaryotic cells has broad applicability both as an assay for the uptake of nucleic acids into cells independent of transcriptional activity and as a tool to study eukaryotic mRNA translation.

Abstract

Mutator stocks of maize produce mutants at many loci at rates 20- to 50-fold above spontaneous levels. Current evidence suggests that this high mutation rate is mediated by an active transposable element system, Mu. Members of this transposable element family are found in approximately 10-60 copies in Mutator stocks. We report here an initial characterization of previously undetected sequences homologous to Mu elements in eight non-Mutator inbred lines and varieties of maize that have a normal low mutation rate. All stocks have approximately 40 copies of sequences homologous only to the terminal repeat and show weak homology to an internal probe. In addition, several of the stocks contain an intact Mu element. One intact Mu element and two terminal-specific clones have been isolated from one non-Mutator line, B37. The cloned sequences have been used to demonstrate that in genomic DNA the intact element, termed Mu1.4B37, is modified, such that restriction sites in its termini are not accessible to cleavage by the HinfI restriction enzyme. This modification is similar to that observed in Mutator lines that have lost activity. We hypothesize that the DNA modification of the Mu-like element may contribute to the lack of Mutator activity in B37.

Abstract

Genomic diversity within the species Zea mays has been examined by measuring the variation in the repetitive component of the nuclear genome among North American inbred lines and varieties. This was done by preparing a set of clones of repetitive maize sequences that differ in function, molecular arrangement and multiplicity and then using these as probes for quantitative hybridization to DNA from various maize genotypes. The comparison showed that the majority of repeated sequences are markedly variable in copy number among the ten maize strains tested. The clone sample contained the rDNA and 5S genes, the major repeat of the chromosome knobs, sequences functioning as origins of DNA replication in yeast (ARS sequences) and randomly cloned sequences of unknown function and chromosomal location. The sequences ranged in reiteration frequency from 200 to greater than 10(5) copies and included both tandemly arrayed and dispersed repeats. The copy numbers were measured by hybridizing labeled cloned sequences to aliquots of high molecular weight genomic DNA that were applied to nitrocellulose filters through a slotted template (slot blotting). The hybridization signal on an autoradiogram occurred in a narrow band that could be scored reliably with a densitometer. This provided a rapid method of determining the abundance of particular repeated sequences in individual plants and plant populations. Using this technique, we found that the copy number of repeated sequences of all types generally varied among the strains by two- to threefold, although at least one sequence showed no detectable variation. In contrast to the variability found between strains, individuals within an inbred line or variety were found to be indistinguishable in terms of specific sequence multiplicity. Each genotype has a different pattern of copy numbers for the set of repeated sequence clones, and this pattern is characteristic of all individuals of a particular genotype. The data also show that the copy number of each sequence varies independently. No strains had uniformly high or low copy numbers for the entire set of probes.

Abstract

The linear, 6397-base pair (bp), mitochondrial S-1 DNA molecule from maize contains a 420-bp segment that is homologous with the chloroplast gene (psbA) that codes for the quinone binding protein of photosystem II. This is the first report of a chloroplast sequence in a naturally occurring viral-like or plasmid DNA. The complete sequence of the S-1 chloroplast segment has been compared with homologous regions of six different chloroplast genes. The S-1 segment has diverged from the other genes both by length mutation and base substitution. Several of the length mutations are exact adjacent tandem duplications of 4 and 5 bp similar to "footprints" left after excision of transposable elements in maize nuclear DNA.

DNA MODIFICATION OF A MAIZE TRANSPOSABLE ELEMENT CORRELATES WITH LOSS OF ACTIVITYPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAChandler, V. L., Walbot, V.1986; 83 (6): 1767-1771

Abstract

An unstable allele of the bronze 2 (bz2) locus was isolated from a Robertson's Mutator Zea mays line containing a family of active transposable Mu elements. This mutation is somatically unstable, resulting in numerous revertant purple sectors on a bronze kernel. By following the variegated kernel phenotype through two generations, several lineages have been identified that have a distorted transmission of the mutant phenotype (fewer variegated kernels are produced than expected). Southern blot analysis of Mu elements in these plants demonstrates a correlation between an inhibition of digestion of Mu elements by certain restriction enzymes and the loss of somatic reversion at the mutant allele. The DNA modification can occur in all the Mu elements in a plant within one generation; however, plants have been identified that contain both modified and unmodified elements, suggesting that the modification can occur in a progressive manner. We hypothesize that the DNA modification results in nonfunctional elements.

Abstract

The graminaceous monocots, including the economically important cereals, seem to be refractory to infection by Agrobacterium tumefaciens, a natural gene transfer system that has been successfully exploited for transferring foreign genes into higher plants. Therefore, direct transfer techniques that are potentially applicable to all plant species have been developed using a few dicot and monocot species as model systems. One of these techniques, electroporation, uses electrical pulses of high field strength to permeabilize cell membranes reversibly so as to facilitate the transfer of DNA into cells. Electroporation-mediated gene transfer has resulted in stably transformed animal cells and transient gene expression in monocot and dicot plant cells. Here we report that electroporation-mediated DNA transfer of a chimaeric gene encoding neomycin phosphotransferase results in stably transformed maize cells that are resistant to kanamycin.

Abstract

We have developed an in organelle DNA synthesis system using isolated mitochondria from maize. The organelles used in this assay are shown to be intact by a number of criteria. Both the high molecular weight components and the smaller plasmid-like components of the mitochondrial genome are used as templates; however, the plasmid-like elements are relatively more active as templates. The termini of the linear plasmids--S-1, S-2 and the 2.3 kbp plasmids--are more highly labelled than internal regions, probably as a result of filling in of gaps at the termini mediated by a DNA polymerase or to exonuclease degradation of the 3' OH termini, with subsequent filling in. Although most of the DNA synthesis observed in this system most likely results from this type of synthesis at DNA nicks or termini, a small amount of specific, potentially replication-associated, synthesis is also detected.

Abstract

A linear 2.3 kb DNA molecule found in maize mitochondria was cloned into pUC8. A natural deletion of this plasmid, found in cmsT and some N (fertile) types of maize plants, was mapped to one end of the plasmid. A minor sequence homology to S-2, another linear mitochondrial plasmid, was detected, as well as more significant sequence homology with chloroplast and maize nuclear DNA. Hybridization to teosinte mitochondrial DNA (mtDNA) revealed the presence of part of the maize plasmid in the high molecular weight mtDNA of the maize relatives. RNA dot hybridization indicates that the plasmid is transcribed in mitochondria. The termini of the 2.3 kb linear plasmid contain inverted repeated sequences; of the first 17 nucleotides of the termini, 16 are identical to the terminal inverted repeats of the linear S plasmids found in the mitochondria of cmsS maize plants.

EXPRESSION OF GENES TRANSFERRED INTO MONOCOT AND DICOT PLANT-CELLS BY ELECTROPORATIONPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAFromm, M., Taylor, L. P., Walbot, V.1985; 82 (17): 5824-5828

Abstract

We have developed a general method for electrically introducing DNA into plant cells. Gene transfer occurs when a high-voltage electric pulse is applied to a solution containing protoplasts and DNA. Carrot protoplasts were used as a model system to optimize gene-transfer efficiency, which was measured 24-48 hr after electroporation by the amount of chloramphenicol acetyltransferase activity resulting from the expression of the introduced chimeric plasmids. Gene-transfer efficiency increased with the DNA concentration and was affected by the amplitude and duration of the electric pulse as well as by the composition of the electroporation medium. Our optimized gene-transfer conditions were effective when applied to tobacco and maize protoplasts, demonstrating that the method is applicable to both monocot and dicot protoplasts.

Abstract

Insertion of the maize transposable element Mu-1 into the first intron of the alcohol dehydrogenase locus (Adh1) of maize produced mutant Adh1-S3034 with 40% of the wild-type level of protein and mRNA. Continued instability at this locus resulted in secondary mutations with lower levels of protein expression. One of these, Adh1-S3034a, has no detectable ADH1 expression. This paper describes the precise nature of the changes in the Adh1 gene that gave rise to the S3034a allele. The Mu-1 element is still present in the mutant, but Adh1 sequences immediately adjacent to the element are deleted. The deletion starts precisely at the Mu-1 insertion site and extends 74 bp leftward removing part of the first intron, the intron:exon junction and 2 bp of the eleventh amino acid codon in the first exon of the gene. Tests for reversion within the somatic tissue of plants show that mutant S3034a, unlike its progenitor, is stably null for ADH1 activity.

CYTOPLASMIC ACIDOSIS AS A DETERMINANT OF FLOODING INTOLERANCE IN PLANTSPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCESRoberts, J. K., Callis, J., Jardetzky, O., Walbot, V., Freeling, M.1984; 81 (19): 6029-6033

Abstract

We present evidence that cytoplasmic acidosis is a cause of meristematic death in hypoxic root tips of maize and pea seedlings. Usually, leakage of acid from the vacuole is responsible for cytoplasmic acidosis. Leakage of acid, which occurs earlier during hypoxia in pea root tips than in maize root tips, appears to account for the lower tolerance of peas for hypoxia. Cytoplasmic acidosis is accelerated in maize root tips that are either (i) deficient in alcohol dehydrogenase, so that lactic acid production continues throughout hypoxia, or (ii) exposed to external CO2 during hypoxia, or (iii) perfused slowly so that escape of CO2 produced during ethanolic fermentation is retarded. All three conditions decrease the length of time maize root tips can tolerate hypoxia; more rapid cytoplasmic acidosis is associated with more rapid death under hypoxia. Possible mechanisms by which cytoplasmic acidosis leads to death are suggested; the mechanism does not involve inhibition of glycolysis by low pH.

MECHANISM OF CYTOPLASMIC PH REGULATION IN HYPOXIC MAIZE ROOT-TIPS AND ITS ROLE IN SURVIVAL UNDER HYPOXIAPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCESRoberts, J. K., Callis, J., Wemmer, D., Walbot, V., Jardetzky, O.1984; 81 (11): 3379-3383

Abstract

We show that a transient lactic fermentation provides the signal triggering ethanol production in hypoxic maize root tips. The signal is cytoplasmic pH. This interaction between lactic and ethanolic fermentation permits tight cytoplasmic pH regulation during hypoxia--cytoplasmic pH remaining near neutrality for several hours. Mutant roots unable to synthesize ethanol can neither regulate cytoplasmic pH nor maintain ATP levels during extended periods of hypoxia and, like vertebrate tissues, are less tolerant of hypoxia than normal maize. This indicates that cytoplasmic pH regulation is an important factor in survival under hypoxia.

Abstract

High resolution gel electrophoresis has allowed the assignment of fragment number and molecular weight to EcoRI, SalI and PstI restriction fragments of mitochondrial DNA from B37 normal (N) and B37 T, C and S male sterile cytoplasmic types of maize. A minimum complexity of 450-475 kb has been established. Hybridization of cloned EcoRI fragments to restriction digests of total mitochondrial DNA suggests that at least 80% of the genome is composed of unique sequences. Restriction fragments of identical size in N, T, C and S contain similar sequence information as evidenced by their hybridization behavior.-The total SalI digest and the larger PstI fragments representing 80% of the total complexity were used to calculate the fraction of shared fragments of each pairwise combination of cytoplasmic types. The C type mtDNA is most closely allied with the other mtDNAs and shares 67% of fragments with S, 65% with N, and 60% with T. The S type mtDNA is quite divergent from N (53% shared fragments) and T (56% shared fragments). N and T share 59% of the fragments. These results are discussed in terms of the origin of mtDNA diversity in maize.